ABSTRACT
DDX3 is a DEAD box RNA helicase with oncogenic properties. RK-33 is developed as a small-molecule inhibitor of DDX3 and showed potent radiosensitizing activity in preclinical tumor models. This study aimed to assess DDX3 as a target in breast cancer and to elucidate how RK-33 exerts its anti-neoplastic effects. High DDX3 expression was present in 35% of breast cancer patient samples and correlated with markers of aggressiveness and shorter survival. With a quantitative proteomics approach, we identified proteins involved in the mitochondrial translation and respiratory electron transport pathways to be significantly downregulated after RK-33 or DDX3 knockdown. DDX3 localized to the mitochondria and DDX3 inhibition with RK-33 reduced mitochondrial translation. As a consequence, oxygen consumption rates and intracellular ATP concentrations decreased and reactive oxygen species (ROS) increased. RK-33 antagonized the increase in oxygen consumption and ATP production observed after exposure to ionizing radiation and reduced DNA repair. Overall, we conclude that DDX3 inhibition with RK-33 causes radiosensitization in breast cancer through inhibition of mitochondrial translation, which results in reduced oxidative phosphorylation capacity and increased ROS levels, culminating in a bioenergetic catastrophe.
Subject(s)
Breast Neoplasms/pathology , DEAD-box RNA Helicases/metabolism , Mitochondria/metabolism , Protein Biosynthesis/drug effects , Radiation-Sensitizing Agents/pharmacology , Azepines/pharmacology , Azepines/therapeutic use , Breast/pathology , Breast Neoplasms/mortality , Breast Neoplasms/therapy , Cell Line, Tumor , DEAD-box RNA Helicases/antagonists & inhibitors , DEAD-box RNA Helicases/genetics , Down-Regulation , Female , Gene Knockdown Techniques , Humans , Imidazoles/pharmacology , Imidazoles/therapeutic use , Mitochondria/drug effects , Mitochondria/genetics , Mitochondria/radiation effects , Oncogenes/drug effects , Proteomics , Radiation-Sensitizing Agents/therapeutic use , Reactive Oxygen Species/metabolism , Reactive Oxygen Species/radiation effects , Survival AnalysisSubject(s)
Carcinoma, Squamous Cell/genetics , DEAD-box RNA Helicases/genetics , Mouth Neoplasms/genetics , Female , Humans , MaleABSTRACT
BACKGROUND: Preoperative CT-angiography (CTA) has shown to reduce operative time in deep inferior epigastric perforator (DIEP) flap breast reconstruction compared to Doppler ultrasonography (US). Although decreased flap loss has been suggested, statistical significant reduction remains indeterminate. The purpose of this review is to evaluate flap loss after preoperative CTA and Doppler US in DIEP-flap breast reconstruction. METHODS: A systematic literature search was performed in MEDLINE, EMBASE, and Cochrane libraries. All articles comparing CTA to Doppler US were selected and critically appraised; data on flap loss were extracted. RESULTS: From 678 studies, eight were selected for appraisal. Six case-control studies were included in the final analysis. Pooled analysis showed CTA resulted in a significant reduction in partial necrosis (odds ratio/OR 0.15; 95% confidence interval/CI 0.07-0.32, P < 0.0001) and decreased flap loss (OR 0.28; 95% CI 0.10-0.79, P = 0.02). CONCLUSIONS: Studies included in this meta-analysis have several limitations. However, most studies find a large clinical advantage of CTA over Doppler US, which reaches statistical significance when combined. As results show that CTA prior to DIEP flap breast reconstruction offers significant clinical benefits, we suggest the routine use of preoperative CTA.
Subject(s)
Angiography/methods , Mammaplasty/methods , Perforator Flap/blood supply , Tomography, X-Ray Computed , Humans , Necrosis , Perforator Flap/pathology , Preoperative Period , Treatment Outcome , Ultrasonography, Doppler , Ultrasonography, MammaryABSTRACT
Heat shock protein (HSP)72, the inducible form of HSP70, protects cells against a variety of injuries, but underlying mechanisms are poorly defined. To investigate the protective effects of HSP72, multiple clones expressing wild-type (WT) HSP72 and two mutants with defective nucleolar and nuclear localization (M45 and 985A, respectively) were made with the tet-off system in C2C12 cells. Four different parameters of cell function/injury were examined after simulated ischemia: protein synthesis, polysome formation, DNA synthesis, and lactate dehydrogenase (LDH release). Overexpression of WT HSP72 was also compared to nontransfected C2C12 cells. As expected, overexpression of HSP72 protected against simulated ischemia and reoxygenation for all parameters. In contrast, both M45 and 985A showed abnormal protein synthesis and polysome formation, both after simulated ischemia and under control conditions. Total RNA was slightly reduced in M45 and 985A at baseline, but 1 h after hypoxia, RNA levels were protected in all clones but significantly decreased in nontransfected C2C12 cells. Clones expressing 985A had nuclear retention of mRNA, suggesting that HSP72 is needed for nuclear export of RNA. All clones, both WT and mutant, had protection of DNA synthesis compared to C2C12 cells, but 985A had greater release of LDH after injury than any other group. These results support a multifactoral protective effect of HSP72, some aspects dependent on nuclear localization with stress and some not. The protection of protein synthesis and polysome formation, and abnormalities in these with the mutants, support a role for HSP72 in these processes both in the normal cell and in injury.
Subject(s)
HSP72 Heat-Shock Proteins/metabolism , Heat-Shock Response/physiology , Muscle, Skeletal/physiology , Myoblasts/physiology , Protein Biosynthesis/physiology , RNA/metabolism , Animals , Cell Hypoxia/physiology , Cell Line , HSP72 Heat-Shock Proteins/chemistry , HSP72 Heat-Shock Proteins/genetics , Mice , Mutagenesis, Site-Directed , Polyribosomes/physiology , Structure-Activity RelationshipABSTRACT
The heat shock proteins (HSPs) are an important family of endogenous, protective proteins that are found in all tissues. In the heart, HSP72, the inducible form of HSP70, has been the most intensely studied. It is well established that HSP72 is induced with ischemia and is cardioprotective. Overexpression of other HSPs also is protective against cardiac injury. Recently, we observed that 17beta-estradiol increases levels of HSPs in male rat cardiac myocytes. We hypothesized that there were gender differences in HSP72 expression in the heart secondary to estrogen. To test this hypothesis, we examined cardiac levels of HSP72 by ELISA in male and female Sprague-Dawley rats. In addition, three other HSPs were assessed by Western blot (HSP27, HSP60, and HSP90). To determine whether estrogen status affected HSP72 expression in other muscles or tissues, two other muscle tissues, slow twitch muscle (soleus muscle) and fast twitch muscle (gastrocnemius muscle), were studied as well as two other organs, the kidney and liver. Because HSP72 is cardioprotective, and females are known to have less cardiovascular disease premenopause, the effects of ovariectomy were examined. We report that female Sprague-Dawley rat hearts have twice as much HSP72 as male hearts. Ovariectomy reduced the level of HSP72 in female hearts, and this could be prevented by estrogen replacement therapy. These data show that the expression of cardiac HSP72 is greater in female rats than in male rats, due to upregulation by estrogen.
