PMID- 30166100 OWN - NLM STAT- MEDLINE DCOM- 20181004 LR - 20220321 IS - 1618-095X (Electronic) IS - 0944-7113 (Linking) VI - 47 DP - 2018 Aug 1 TI - Xanthone-rich extract from Gentiana dinarica transformed roots and its active component norswertianin induce autophagy and ROS-dependent differentiation of human glioblastoma cell line. PG - 151-160 LID - S0944-7113(18)30087-4 [pii] LID - 10.1016/j.phymed.2018.03.052 [doi] AB - BACKGROUND: Glioblastoma multiforme (GMB) is the most malignant of all brain tumors with poor prognosis. Anticancer potential of xanthones, bioactive compounds found in Gentiana dinarica, is well-documented. Transformation of G. dinarica roots with Agrobacterium rhizogenes provides higher xanthones accumulation, which enables better exploitation of these anticancer compounds. HYPOTHESIS/PURPOSE: The aim of this study was to investigate antiglioma effect of three different G. dinarica extracts: E1-derived from untransformed roots, E2-derived from roots transformed using A. rhizogenes strain A4M70GUS, and E3-derived from roots transformed using A. rhizogenes strain 15834/PI. Further, mechanisms involved in anticancer potential of the most potent extract were examined in detail, and its active component was determined. METHODS: The cell viability was assessed using MTT and crystal violet test. Cell cycle analysis, the expression of differentiation markers, the levels of autophagy, and oxidative stress were analyzed by flow cytometry. Autophagy and related signaling pathways were assessed by immunoblotting. RESULTS: E3, in contrast to E1 and E2, strongly reduced growth of U251 human glioblastoma cells, triggered cell cycle arrest in G(2)/M phase, changed cellular morphology, and increased expression of markers of differentiated astrocytes (glial fibrillary acidic protein) and neurons (beta-tubulin). E3 stimulated autophagy, as demonstrated by enhanced intracellular acidification, increased microtubule-associated light chain 3B (LC3-I) conversion to autophagosome associated LC3-II, and decreased level of selective autophagy target p62. Induction of autophagy was associated with Akt-dependent inhibition of main autophagy suppressor mammalian target of rapamycin (mTOR). Both genetic and pharmacological inhibition of autophagy suppressed the expression of differentiation markers, but had no effect on cell cycle arrest in E3-treated cells. E3 stimulated oxidative stress, and antioxidants vitamin E and N-acetyl cysteine inhibited autophagy and differentiation of E3-treated U251 cells. The most prevalent compound of E3, xanthone aglycone norswertianin, also arrested glioblastoma cell proliferation in G(2)/M phase and induced glioblastoma cell differentiation through induction of autophagy and oxidative stress. CONCLUSION: These results indicate that E3 and its main active component norswertianin may serve as a potential candidate for differentiation therapy of glioblastoma. CI - Copyright (c) 2018 Elsevier GmbH. All rights reserved. FAU - Tovilovic-Kovacevic, Gordana AU - Tovilovic-Kovacevic G AD - Department of Biochemistry, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Despot Stefan Blvd 142, 11000 Belgrade, Serbia. FAU - Krstic-Milosevic, Dijana AU - Krstic-Milosevic D AD - Department of Plant Physiology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Despot Stefan Blvd 142, 11000 Belgrade, Serbia. FAU - Vinterhalter, Branka AU - Vinterhalter B AD - Department of Plant Physiology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Despot Stefan Blvd 142, 11000 Belgrade, Serbia. FAU - Toljic, Mina AU - Toljic M AD - Genetic Laboratory Department, Obstetrics and Gynecology Clinic "Narodni Front", Street Kraljice Natalije 62, 11000 Belgrade, Serbia. FAU - Perovic, Vladimir AU - Perovic V AD - Institute for Microbiology and Immunology, School of Medicine, University of Belgrade, Dr Subotic Street 1, 11000 Belgrade, Serbia. FAU - Trajkovic, Vladimir AU - Trajkovic V AD - Institute for Microbiology and Immunology, School of Medicine, University of Belgrade, Dr Subotic Street 1, 11000 Belgrade, Serbia. FAU - Harhaji-Trajkovic, Ljubica AU - Harhaji-Trajkovic L AD - Department of Neurophysiology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Despot Stefan Blvd 142, 11000 Belgrade, Serbia. Electronic address: buajk@yahoo.com. FAU - Zogovic, Nevena AU - Zogovic N AD - Department of Neurophysiology, Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Despot Stefan Blvd 142, 11000 Belgrade, Serbia. Electronic address: nevenar@ibiss.bg.ac.rs. LA - eng PT - Journal Article DEP - 20180319 PL - Germany TA - Phytomedicine JT - Phytomedicine : international journal of phytotherapy and phytopharmacology JID - 9438794 RN - 0 (Plant Extracts) RN - 0 (Reactive Oxygen Species) RN - 0 (Xanthones) RN - 54954-12-0 (norswertianolin) RN - EC 2.7.1.1 (MTOR protein, human) RN - EC 2.7.11.1 (TOR Serine-Threonine Kinases) SB - IM MH - Autophagy/*drug effects MH - Brain Neoplasms/pathology MH - Cell Cycle Checkpoints MH - Cell Differentiation/*drug effects MH - Cell Line, Tumor MH - Cell Proliferation/drug effects MH - Cell Survival/drug effects MH - Gentiana/*chemistry MH - Glioblastoma/pathology MH - Humans MH - Oxidative Stress MH - Plant Extracts/pharmacology MH - Plant Roots/chemistry MH - Reactive Oxygen Species/metabolism MH - Signal Transduction/drug effects MH - TOR Serine-Threonine Kinases/metabolism MH - Xanthones/*pharmacology OTO - NOTNLM OT - Autophagy OT - Cell differentiation OT - Gentiana dinarica transformed roots OT - Glioblastoma OT - Norswertianin OT - Xanthone EDAT- 2018/09/01 06:00 MHDA- 2018/10/05 06:00 CRDT- 2018/09/01 06:00 PHST- 2017/11/03 00:00 [received] PHST- 2018/02/07 00:00 [revised] PHST- 2018/03/19 00:00 [accepted] PHST- 2018/09/01 06:00 [entrez] PHST- 2018/09/01 06:00 [pubmed] PHST- 2018/10/05 06:00 [medline] AID - S0944-7113(18)30087-4 [pii] AID - 10.1016/j.phymed.2018.03.052 [doi] PST - ppublish SO - Phytomedicine. 2018 Aug 1;47:151-160. doi: 10.1016/j.phymed.2018.03.052. Epub 2018 Mar 19.