Human Airway Organoids and Multimodal Imaging-Based Toxicity Evaluation of 1-Nitropyrene.

Despite significant advances in understanding the general health impacts of air pollution, the toxic effects of air pollution on cells in the human respiratory tract are still elusive. A robust, biologically relevant in vitro model for recapitulating the physiological response of the human airway is needed to obtain a thorough understanding of the molecular mechanisms of air pollutants. In this study, by using 1-nitropyrene (1-NP) as a proof-of-concept, we demonstrate the effectiveness and reliability of evaluating environmental pollutants in physiologically active human airway organoids. Multimodal imaging tools, including live cell imaging, fluorescence microscopy, and MALDI-mass spectrometry imaging (MSI), were implemented to evaluate the cytotoxicity of 1-NP for airway organoids. In addition, lipidomic alterations upon 1-NP treatment were quantitatively analyzed by nontargeted lipidomics. 1-NP exposure was found to be associated with the overproduction of reactive oxygen species (ROS), and dysregulation of lipid pathways, including the SM-Cer conversion, as well as cardiolipin in our organoids. Compared with that of cell lines, a higher tolerance of 1-NP toxicity was observed in the human airway organoids, which might reflect a more physiologically relevant response in the native airway epithelium. Collectively, we have established a novel system for evaluating and investigating molecular mechanisms of environmental pollutants in the human airways via the combinatory use of human airway organoids, multimodal imaging analysis, and MS-based analyses.

Phase 1 Trial of SBRT to the Prostate Fossa After Prostatectomy.

PURPOSE: The primary objective was to evaluate the maximum tolerated dose (within 10 weeks after treatment) associated with increasing hypofractionation to the prostate fossa (PF). We hypothesized that escalating the dose per fraction (fx) to the PF would have acceptable toxicity. MATERIALS AND METHODS: Tested dose levels (DLs) were 3.6 Gy × 15 fx (DL1); 4.7 Gy × 10 fx (DL2); and 7.1 Gy × 5 fx (DL3). Escalation followed a 6 + 6 rules-based design with 12 patients required at the maximum tolerated dose. Dose-limiting toxicity was defined as grade (G) ≥3, gastrointestinal (GI) or genitourinary (GU) toxicity by National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.03). Patients completed quality-of-life questionnaires. RESULTS: Twenty-four patients with indications for adjuvant or salvage radiation therapy (RT) enrolled (6 at DL1 and 2; 12 at DL3). All patients had at least 6 months of follow-up (median follow-up, 14.1 months). Four patients received concurrent androgen deprivation therapy. No G ≥ 3 GI or GU toxicity was seen at any DL; 2 of 6 patients in the DL1 group, 3 of 6 in DL2, and 7 of 12 in DL3 experienced G2 GI toxicity during RT. Except in 1 patient, all acute G2 GI toxicity resolved by 10 weeks. Three of 12 patients reported an increase to G1 and G2 GU toxicity in the 2 weeks after RT in groups DL1 and DL2 and 1 of 12 patients in DL3. At week 2 after RT, decline in the 26-item Expanded Prostate Cancer Index Composite bowel domain met criteria for a minimally important difference in 71% of patients. At week 10, 1 of 6, 2 of 6, and 7 of 11 patients at DLs 1, 2, and 3, respectively, still met minimally important difference criteria. International Prostate Symptom Scores worsened 2 weeks after treatment but improved by 6 to 10 weeks. CONCLUSIONS: Dose escalation up to 7.1 Gy × 5 fx to the PF was completed without acute G ≥ 3 toxicity. There was transient G2 rectal toxicity at all DLs during and immediately after RT. We must perform long-term follow-up and assessment of late toxicity of SBRT to the PF.

Novel Treatment Setup for Urethral Cancer: Use of the Prone Technique for Radiotherapy of the Penis.

Urethral cancer (UC) is a rare malignancy with a poor prognosis. Since local recurrence is common and associated with morbidity, case series have reported on the use of adjuvant radiotherapy. Radiotherapy treatment setup for malignancies of the penis can be challenging because of variability in anatomic positioning. This variability can lead to lack of reproducibility. We propose a novel external beam radiotherapy technique for the treatment of UC: prone positioning. This technique has been used to treat breast cancers successfully and can be used to treat any variety of penile malignancies. We present 2 patients who were treated using this positioning.

Fragmentation of the Golgi apparatus: an early apoptotic event independent of the cytoskeleton.

The Golgi apparatus undergoes irreversible fragmentation during apoptosis, in part as a result of caspase-mediated cleavage of several Golgi-associated proteins. However, Golgi structure and orientation is also regulated by the cytoskeleton and cytoskeletal changes have been implicated in inducing apoptosis. Consequently, we have analyzed the role of actin filaments and microtubules in apoptotic Golgi fragmentation. We demonstrate that in Fas receptor-activated cells, fragmentation of the Golgi apparatus was an early event that coincided with release of cytochrome c from mitochondria. Significantly, Golgi fragmentation preceded major changes in the organization of both the actin cytoskeleton and microtubules. In staurosporine-treated cells, actin filament organization was rapidly disrupted; however, the Golgi apparatus maintained its juxtanuclear localization and underwent complete fragmentation only at later times. Attempts to stabilize actin filaments with jasplakinolide prior to treatment with staurosporine did not prevent Golgi fragmentation. Finally, in response to Fas receptor activation or staurosporine treatment the levels of beta-actin or alpha-tubulin remained unaltered, whereas several Golgi proteins, p115 and golgin-160, underwent caspase-mediated cleavage. Our data demonstrate that breakdown of the Golgi apparatus is an early event during apoptosis that occurs independently of major changes to the actin and tubulin cytoskeleton.

A caspase cleavage fragment of p115 induces fragmentation of the Golgi apparatus and apoptosis.

In mammalian cells, the Golgi apparatus undergoes extensive fragmentation during apoptosis. p115 is a key vesicle tethering protein required for maintaining the structural organization of the Golgi apparatus. Here, we demonstrate that p115 was cleaved during apoptosis by caspases 3 and 8. Compared with control cells expressing native p115, those expressing a cleavage-resistant form of p115 delayed Golgi fragmentation during apoptosis. Expression of cDNAs encoding full-length or an NH2-terminal caspase cleavage fragment of p115 had no effect on Golgi morphology. In contrast, expression of the COOH-terminal caspase cleavage product of p115 itself caused Golgi fragmentation. Furthermore, this fragment translocated to the nucleus and its expression was sufficient to induce apoptosis. Most significantly, in vivo expression of the COOH-terminal fragment in the presence of caspase inhibitors, or upon coexpression with a cleavage-resistant mutant of p115, showed that p115 degradation plays a key role in amplifying the apoptotic response independently of Golgi fragmentation.