2-Deoxyglucose combined with wild-type p53 overexpression enhances cytotoxicity in human prostate cancer cells via oxidative stress

Overexpression of the tumor suppressor gene, wild-type p53 (wtp53), using adenoviral vectors (Adp53) has been suggested to kill cancer cells by hydroperoxide-mediated oxidative stress [1,2] and nutrient distress induced by the glucose analog, 2-deoxyglucose (2DG), has been suggested to enhance tumor cell killing by agents that induce oxidative stress via disrupting hydroperoxide metabolism [3,4]. In the current study clonogenic cell killing of PC-3 and DU-145 human prostate cancer cells (lacking functional p53) mediated by 4 h exposure to 50 plaque forming units (pfus)/cell of Adp53 (that caused the enforced overexpression of wtp53) was significantly enhanced by treatment with 2DG. Accumulation of glutathione disulfide was found to be significantly greater in both cell lines treated with 2DG+Adp53 and both cell lines treated with 2DG+Adp53 showed a approximately 2-fold increases in dihydroethidine (DHE) and 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (CDCFH(2)) oxidation, indicative of increased steady-state levels of O(2)(.-) and hydroperoxides, respectively. Finally, overexpression of catalase or glutathione peroxidase using adenoviral vectors partially, but significantly, protected DU-145 cells from the toxicity induced by 2DG+Adp53 treatment. These results show that treatment of human prostate cancer cells with the combination of 2DG (a nutrient stress) and overexpression of the tumor suppressor gene, wtp53, enhances clonogenic cell killing by a mechanism that involves oxidative stress as well as allowing for the speculation that inhibitors of glucose and hydroperoxide metabolism can be used in combination with Adp53 gene therapy to enhance therapeutic responses.     

DNAH6 and Its Interactions with PCD Genes in Heterotaxy and Primary Ciliary Dyskinesia

Heterotaxy, a birth defect involving left-right patterning defects, and primary ciliary dyskinesia (PCD), a sinopulmonary disease with dyskinetic/immotile cilia in the airway are seemingly disparate diseases. However, they have an overlapping genetic etiology involving mutations in cilia genes, a reflection of the common requirement for motile cilia in left-right patterning and airway clearance. While PCD is a monogenic recessive disorder, heterotaxy has a more complex, largely non-monogenic etiology. In this study, we show mutations in the novel dynein gene DNAH6 can cause heterotaxy and ciliary dysfunction similar to PCD. We provide the first evidence that trans-heterozygous interactions between DNAH6 and other PCD genes potentially can cause heterotaxy. DNAH6 was initially identified as a candidate heterotaxy/PCD gene by filtering exome-sequencing data from 25 heterotaxy patients stratified by whether they have airway motile cilia defects. dnah6 morpholino knockdown in zebrafish disrupted motile cilia in Kupffer’s vesicle required for left-right patterning and caused heterotaxy with abnormal cardiac/gut looping. Similarly DNAH6 shRNA knockdown disrupted motile cilia in human and mouse respiratory epithelia. Notably a heterotaxy patient harboring heterozygous DNAH6 mutation was identified to also carry a rare heterozygous PCD-causing DNAI1 mutation, suggesting a DNAH6/DNAI1 trans-heterozygous interaction. Furthermore, sequencing of 149 additional heterotaxy patients showed 5 of 6 patients with heterozygous DNAH6 mutations also had heterozygous mutations in DNAH5 or other PCD genes. We functionally assayed for DNAH6/DNAH5 and DNAH6/DNAI1 trans-heterozygous interactions using subthreshold double-morpholino knockdown in zebrafish and showed this caused heterotaxy. Similarly, subthreshold siRNA knockdown of Dnah6 in heterozygous Dnah5 or Dnai1 mutant mouse respiratory epithelia disrupted motile cilia function. Together, these findings support an oligogenic disease model with broad relevance for further interrogating the genetic etiology of human ciliopathies.     

Production of cinnamyl alcohol glycosides by biotransformation in roseroot callus cells

Plant Cell, Tissue and Organ Culture (PCTOC) - Decalepis salicifolia (Bedd. ex Hook.f.) Venter is a new source of the commercially important flavor compound 2-hydroxy-4-methoxybenzaldehyde (2H4MB),...     

When reverse genetics meets physiology: the use of site-specific recombinases in mice

The use of site-specific recombinases enables the precise introduction of defined genetic mutations into the mouse genome. In theory, any deletion, point mutation, inversion or translocation can be modeled in mice. Because gene targeting is controlled both spatially and temporally, the function of a given gene can be studied in the desired cell types and at a specific time point. This 'genetic dissection' allows to define gene function in development, physiology or behavior. In this review, we focus on the technical possibilities of Cre and other site-specific recombinases but also discuss their limitations.     

Iron and Porphyrin Trafficking in Heme Biogenesis

Iron is an essential element for diverse biological functions. In mammals, the majority of iron is enclosed within a single prosthetic group: heme. In metazoans, heme is synthesized via a highly conserved and coordinated pathway within the mitochondria. However, iron is acquired from the environment and subsequently assimilated into various cellular pathways, including heme synthesis. Both iron and heme are toxic but essential cofactors. How is iron transported from the extracellular milieu to the mitochondria? How are heme and heme intermediates coordinated with iron transport? Although recent studies have answered some questions, several pieces of this intriguing puzzle remain unsolved.     

Comparison of tyrosinase levels in amelanotic and melanotic melanoma cell cultures by a competitive enzyme-linked immunoadsorbent assay and by immunotitration analysis

Melanogenesis in mammalian pigment cells is regulated by changes in the activity of tyrosinase, the rate-limiting enzyme for melanin synthesis. Because recent evidence suggests that this enzyme may exist in pigment cells in both active and inactive stages, a competitive enzyme-linked immunoadsorbent assay (ELISA) was developed to compare tyrosinase levels in amelanotic and melanotic melanoma cell clones. The melanotic cell line used for this study, MEL-11A, had basal tyrosinase levels approximately 40 times that of the amelanotic cell line, AM-7. Both cell lines responded to melanocyte-stimulating hormone by demonstrating large increases in tyrosinase activity. For competitive ELISA analysis of tyrosinase levels in these two clones, microtiter plates were coated with purified tyrosinase, and trypsinized cell extracts were tested for their ability to compete with bound tyrosinase for antibody binding. Although tyrosinase activity in the amelanotic clone was 1/40 that of the melanotic clone, immunoreactive tyrosinase levels in AM-7 cells were found to be approximately one-half that present in the melanotic clone. Additional evidence for the presence of an inactive (or at least, catalytically less active) enzyme in AM-7 cells was obtained from immunotitration analysis of tyrosinase in cell extracts from both cell lines. These results suggest that at least some amelanotic melanoma cells may contain significant levels of catalytically inactive tyrosinase molecules and that the level of pigmentation in mammalian melanocytes may be regulated by a tyrosinase activation process.     

An SAR study of hydroxy-trifluoromethylpyrazolines as inhibitors of Orai1-mediated store operated Ca2+ entry in MDA-MB-231 breast cancer cells using a convenient Fluorescence Imaging Plate Reader assay

The proteins Orai1 and STIM1 control store-operated Ca²⁺ entry (SOCE) into cells. SOCE is important for migration, invasion and metastasis of MDA-MB-231 human triple negative breast cancer (TNBC) cells and has been proposed as a target for cancer drug discovery. Two hit compounds from a medium throughput screen, displayed encouraging inhibition of SOCE in MDA-MB-231 cells, as measured by a Fluorescence Imaging Plate Reader (FLIPR) Ca²⁺ assay. Following NMR spectroscopic analysis of these hits and reassignment of their structures as 5-hydroxy-5-trifluoromethylpyrazolines, a series of analogues was prepared via thermal condensation reactions between substituted acylhydrazones and trifluoromethyl 1,3-dicarbonyl arenes. Structure-activity relationship (SAR) studies showed that small lipophilic substituents at the 2- and 3-positions of the RHS and 2-, 3- and 4-postions of the LHS terminal benzene rings improved activity, resulting in a novel class of potent and selective inhibitors of SOCE.     

<Emphasis Type="Italic">Rhodiola rosea</Emphasis> L.: from golden root to green cell factories

Rhodiola rosea L. is a worldwide popular plant with adaptogenic activities that have been and currently are exploited in the traditional medicine of many countries, as well as, examined in a number of clinical trials. More than 140 chemical structures have been identified which belong to several natural product classes, including phenylpropanoid glycosides, phenylethanoids, flavonoids and essential oils, and are mainly stored in the rhizomes and the roots of the plant. A number of mechanisms contribute to the adaptogenic activities of R. rosea preparations and its phytochemical constituents. Among them, the intrinsic inducible mammalian stress responses and their effector proteins, such as heat shock protein 70 (Hsp70), are the most prominent. Due to its popular medicinal use, which has led to depletion of its natural habitats, R. rosea is now considered as endangered in most parts of the world. Conservation, cultivation and micropropagation are all implemented as potential preservation strategies. A number of in vitro systems of R. rosea are being developed as sources of pharmaceutically valuable secondary metabolites. These are greatly facilitated by advances in elucidation of the biosynthetic pathways and the enzymes, which catalyse the production of these secondary metabolites in the plant. In addition, biotechnological approaches show promise towards achieving sustainable production of R. rosea secondary metabolites.     

Comparison of high-dose Cisplatin-based chemoradiotherapy and Cetuximab-based bioradiotherapy for p16-positive oropharyngeal squamous cell carcinoma in the context of revised HPV-based staging

Aim: To perform a comparison of Cisplatin vs. Cetuximab in p16-positive oropharyngeal squamous cell carcinoma (OPSCC) in the context of the revised HPV-based staging.Background: Previous reports comparing these agents in head and neck cancer have included heterogenous disease and p16-status.Materials and methods: A retrospective review was conducted from 2006 to 2016 of patients with p16-positive OPSCC who underwent definitive radiotherapy concurrent with either triweekly Cisplatin (n?=?251) or Cetuximab (n?=?40). AJCC 8th Edition staging was adapted.Results: Median follow-up for surviving patients was 40 months. On multivariate analysis for all-comers, comparing Cisplatin and Cetuximab, 3-year locoregional recurrence (LRR): 6% vs. 16% (p?=?0.07), 3-year distant metastasis (DM): 8% vs. 21% (p?=?0.04), 3-year overall recurrence rate (ORR): 11% vs. 29% (p?=?0.01), and 3-year cause-specific survival (CSS): 94% vs. 79% (p?=?0.06), respectively. On stage-based subgroup analysis, for stage III disease, 3-year LRR: 5% vs. 10% (p?=?0.51), 3-year DM: 7% vs. 16% (p?=?0.32), 3-year ORR: 10% vs. 23% (p?=?0.15), and 3-year CSS: 95% vs. 82% (p?=?0.38). For stage III disease, 3-year LRR: 10% vs. 40% (p?=?0.07), 3-year DM: 9% vs. 43% (p?=?0.07), 3-year ORR: 15% vs. 55% (p?=?0.04), and 3-year CSS: 94% vs. 57% (p?=?0.048).Conclusions: When given concurrently with radiotherapy, Cetuximab and triweekly Cisplatin demonstrated comparable efficacy for AJCC 8th Edition stage I–II p16-positive OPSCC. However, Cetuximab appeared to be associated with higher rates of treatment failure and cancer-related deaths in stage III disease. Upon availability of the RTOG 1016 trial results, analysis based on the revised HPV-based staging should be performed to confirm these findings.