MESENCHYMAL STEM CELLS AS THERAPY AGAINST HUMAN GLIOBLASTOMA MULTIFORME

MESENCHYMAL STEM CELLS AS THERAPY AGAINST HUMAN GLIOBLASTOMA MULTIFORME YULYANA (B.Sc (Hons.), UNSW) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2013 ACKNOWLEDGEMENTS Research is not a one-man show, and certainly, this thesis would not have been completed without the support of many, to whom I wish to express my gratitudes First and foremost, I am grateful to Assoc Prof Paula Lam for accepting me in the lab, for the opportunity to undertake this M.Sc project and for your guidance, advice, and patience as my supervisor to bring out the best in me To Dr Ivy Ho, for your endless support, guidance and patience; for your encouragement, prayers and positive thinking during trying moments, I am forever grateful For the many lunches, cups of coffee and chats that we shared, they are invaluable to me And your giggles never fail to cast away the gloomy atmosphere To my great lab members, past and present To Dr Sia Kian Chuan, for sharing your journey, for your many advices, a pair of ears, and your pet fishes, I am forever thankful To Jennifer Newman and Toh Xin Yi, I am grateful for your support and dependable assistance To my dearest friends, near and far Your listening ears, comforting words and a pat on my back are more than I could have asked for Thank you for being there when the going got rough Last but not least, to my precious family, without whom, I will not be here Without your unconditional love and support, I would not have been able to weather the storm You are forever my source of strength and motivation iii TABLE OF CONTENTS Declaration Acknowledgements Table of contents Summary List of Tables List of Figures List of Abbreviations ii iii iv vii ix x xii 1 INTRODUCTION 1.1 Glioblastoma Multiforme 1.2 Treatment options for GBMs 1.2.1 Current standard treatment regime for GBMs 1.2.2 Gene therapy for GBMs 4 1.3 Mesenchymal Stem Cell as delivery vector 1.3.1 Mesenchymal Stem Cell (MSCs) 1.3.2 Tumor tracking properties of MSCs 1.3.3 Genetically-engineered MSCs 7 10 1.4 TRAIL as potent apoptosis inducer 1.4.1 TRAIL and its receptors 1.4.2 TRAIL apoptotic pathway 1.4.3 TRAIL resistance and strategies to overcome resistance 10 10 12 14 1.5 Cell communication and adhesion 1.5.1 Gap junctions 1.5.2 Connexin 43 1.5.3 Adhesion-mediated apoptosis resistance 19 20 21 24 1.6 Hypothesis and Study aims 24  2 MATERIALS AND METHODS 26  2.1 Cell culture and reagents 2.1.1 Glioma spheroid culture 2.1.2 MSCs 2.1.3 Primary glioma cell culture 26 27 27 27 2.2 Cloning of pHGCX-TRAIL Herpes Simplex Virus-1 (HSV-1) Amplicon viral vector 2.2.1 Viral packaging and purification 2.2.2 Virus titering 28 29 29 2.3 Cell transfection and transduction 2.3.1 Standard transfection 2.3.2 RNAi transfection 2.3.3 Viral transduction 30 30 30 31 iv 2.4 MSCs characterization 2.4.1 MSCs differentiation 2.4.2 MSCs surface markers analysis 31 31 32 2.5 Enzyme-linked Immunosorbent Assay (ELISA) 33 2.6 Harvesting of CM 33 2.7 In vitro migration assay 34 2.8 Trypan blue dye exclusion assay 34 2.9 Caspase-3 activity assay 35 2.10 FACS analysis 2.10.1 FACS analysis for eGFP 2.10.2 Surface receptor analysis 2.10.3 Cell cycle analysis 35 35 36 36 2.11 Immunofluorescence staining 36 2.12 Dye transfer assay 37 2.13 Intracranial glioma mouse model 37 2.14 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining 38 2.15 Western blot analysis 39 2.16 RNA isolation 40 2.17 Real Time Reverse Transcriptase Polymerase Chain Reaction 40 2.18 Statistical analysis 41 3 RESULTS 42  3.1 Construction of TRAIL-secreting HSV-1 vector and validation of its functionality 3.1.1 Construction of TRAIL-secreting HSV-1 vector, pHGCX-TRAIL and its functional validation 3.1.2 Functionality of pHGCX-TRAIL in MSCs 3.1.3 Glioma cells response variability to TRAIL 42 42 48 3.2 Physiological effect of CBX 3.2.1 CBX does not affect glioma cells and MSCs viability 3.2.2 CBX blocks GJIC and may affect cell cycle 50 50 54 3.3 CBX enhances TRAIL-induced apoptosis 3.3.1 CBX enhances TRAIL-induced apoptosis in glioma cells 3.3.2 CBX enhances TRAIL-induced apoptosis in patient-derived glioma cultures 3.3.3 Double arm therapy of CBX and MSC-TRAIL prolonged survival of intracranial mouse model 56 56 42 63 64 v 3.4 Mechanisms contributed by CBX in augmenting TRAIL-induced apoptosis 66 3.4.1 CBX downregulates Cx43 66 3.4.2 CBX upregulates DR5 expression 70 4 DISCUSSION 74  4.1 Improvement in therapeutic vector system and its limitation 77 4.2 Cytoplasmic Cx43 may form functional GJ in glioma 83 4.3 GJ-dependent-mediation of cell death 84 4.4 CBX and cellular stress 85 5 FUTURE STUDIES 91 BIBLIOGRAPHY 94 APPENDIX - List of publications 119 vi SUMMARY Glioblastoma multiforme (GBM) is the most common and aggressive brain tumors that to this day are incurable despite the advancement in surgical techniques and standard therapies One contributing factor is the inherent ability of GBMs to disseminate and invade into the normal brain parenchyma, rendering complete removal of tumor cells difficult to achieve The development of anti-glioma gene therapies has become an alternative approach to curb the limitations of standard therapy However, direct administration of gene therapy vectors into brain tumors fails to achieve significant therapeutic efficacy The poor treatment efficacy is attributed to the limited distribution of therapeutic vectors into the brain tumor region, as well as the invasive nature and heterogeneity of GBMs Therefore, improved modalities are needed to effectively circumvent the limitation in the distribution of therapeutics The main objective of this study was to improve the delivery system for GBM treatment by harnessing the tumor-tropic property of human mesenchymal stem cells (MSCs) to deliver therapeutic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) Furthermore, we postulated that the therapeutic efficacy of soluble TRAIL mediated by MSCs could be enhanced when gap junction communication between glioma cells is disrupted To this end, MSCs were transduced with herpes simplex virus-1 amplicon viruses that were engineered to secrete soluble and functional TRAIL (MSC-TRAIL) Carbenoxolone (CBX), a known gap junction inhibitor, was used to interfere with gap junction communication in glioma cells which has been implicated in treatment resistance The therapeutic efficacy of CBX on MSC-TRAILinduced apoptosis was subsequently evaluated in human glioma cell lines, patientderived gliomas, and orthotopic glioma mouse model vii The results in this thesis demonstrated that combination treatment of MSC-TRAIL and CBX significantly enhanced glioma cell death compared to single treatment Enhanced cell death is specific to human gliomas but not normal astrocytes and this included patient-derived isolates that are normally insensitive to TRAIL More importantly, dual arm therapy of MSC-TRAIL and CBX effectively prolonged the survival of orthotopic glioma mice by ~27% when compared with the control mice, indicating that interference of gap junction communication could improve therapeutic efficacy of MSC-TRAIL Molecular evaluation on the mechanisms of enhanced cell death by MSC-TRAIL and CBX showed that it was mediated through an upregulation of C/EBP homologous protein and death receptor expressions Death signals from death receptor were further amplified through the engagement of intrinsic apoptosis pathway and downregulation of anti-apoptotic protein Bcl-2 Furthermore, the results have demonstrated that the downregulation of connexin 43 by CBX further amplified the death signals by preventing these signal molecules to be diluted out and thus sealing the fate of the cells into apoptosis These mechanisms synergistically resulted in the increase in therapeutic efficacy In conclusion, this study has demonstrated that MSC-TRAIL when combined with gap junction inhibitor may serve as an effective therapy against human GBMs It may potentially be applied for clinical use for the following reasons: (1) No obvious physiological or neurological effect was observed in mice administered with CBX; (2) CBX acts synergistically with MSC-TRAIL at multiple levels, which is particularly advantageous as tumor cells employ multiple resistance mechanism to therapeutic agents viii LIST OF TABLES Table 1.1 WHO classification of brain tumors and their features Table 1.2 Compounds used in combinatorial strategies with TRAIL and their mechanism of actions 19 ix LIST OF FIGURES Figure 1.1 Pathological features of malignant gliomas Figure 1.2 Pathways in the development of malignant gliomas Figure 1.3 Glioma tumor tropism of BM-hMSCs Figure 1.4 The TRAIL signaling pathway 13 Figure 1.5 Gap junctions in cell membranes 22 Figure 3.1 Construction and functionality of pHGCX-TRAIL 44 Figure 3.2 Characterization of bone marrow-derived MSCs 45 Figure 3.3 Functionality of pHGCX-TRAIL in MSCs 46 Figure 3.4 Cell surface expression of TRAIL receptors 48 Figure 3.5 Glioma cells response variability to TRAIL 49 Figure 3.6 Effect of CBX on MSCs 52 Figure 3.7 Effect of CBX on glioma cells 53 Figure 3.8 CBX may affect cell cycle progression of glioma cells 55 Figure 3.9 CBX blocks GJIC in glioma cells 56 Figure 3.10 CBX augments MSC-mediated TRAIL-induced apoptosis in human glioma cell lines 57 Figure 3.11 CBX modulates proteins involved in the apoptotic pathway 59 Figure 3.12 CBX augments MSC-mediated TRAIL-induced apoptosis in patient-derived primary glioma cells 62 CBX synergizes with MSC-TRAIL to prolong the survival of glioma-bearing mice 65 Figure 3.14 Connexins expression in glioma cells 67 Figure 3.15 CBX downregulates Cx43 67 Figure 3.16 Downregulation of Cx43 by CBX enhances TRAIL-induced apoptosis in glioma cells 68 Figure 3.13 Figure 3.17 CBX upregulates DR5 expression Figure 3.18 71 Enhanced TRAIL-apoptosis by CBX is partially mediated by x 124 Allensworth, JL, Aird, KM, Aldrich, AJ, Batinic-Haberle, I, and Devi, GR (2012) XIAP inhibition and generation of reactive oxygen species enhances TRAIL sensitivity in inflammatory breast cancer cells Mol Cancer Ther 11: 1518-1527 125 Siegelin, MD, Reuss, DE, Habel, A, Rami, A, and von Deimling, A (2009) Quercetin promotes degradation of survivin and thereby enhances deathreceptor-mediated apoptosis in glioma cells Neuro Oncol 11: 122-131 126 Guo, F, Nimmanapalli, R, Paranawithana, S, Wittman, S, Griffin, D, Bali, P, O'Bryan, E, Fumero, C, Wang, HG, and Bhalla, K (2002) Ectopic overexpression of second mitochondria-derived activator of caspases (Smac/DIABLO) or cotreatment with N-terminus of Smac/DIABLO peptide potentiates epothilone B derivative-(BMS 247550) and Apo-2L/TRAILinduced apoptosis Blood 99: 3419-3426 127 Li, L, Thomas, RM, Suzuki, H, De Brabander, JK, Wang, X, and Harran, PG (2004) A small molecule Smac mimic potentiates TRAIL- and TNFalphamediated cell death Science 305: 1471-1474 128 Wu, MS, Wang, GF, Zhao, ZQ, Liang, Y, Wang, HB, Wu, MY, Min, P, Chen, LZ, Feng, QS, Bei, JX, Zeng, YX, and Yang, D (2013) Smac mimetics in combination with TRAIL selectively target cancer stem cells in nasopharyngeal carcinoma Mol Cancer Ther 12: 1728-1737 129 Zhu, H, Guo, W, Zhang, L, Davis, JJ, Wu, S, Teraishi, F, Cao, X, Smythe, WR, and Fang, B (2005) Enhancing TRAIL-induced apoptosis by Bcl-X(L) siRNA Cancer Biol Ther 4: 393-397 130 Rosato, RR, Almenara, JA, Coe, S, and Grant, S (2007) The multikinase inhibitor sorafenib potentiates TRAIL lethality in human leukemia cells in association with Mcl-1 and cFLIPL down-regulation Cancer Res 67: 94909500 131 Ray, S, Bucur, O, and Almasan, A (2005) Sensitization of prostate carcinoma cells to Apo2L/TRAIL by a Bcl-2 family protein inhibitor Apoptosis 10: 1411-1418 132 Miao, L, Yi, P, Wang, Y, and Wu, M (2003) Etoposide upregulates Baxenhancing tumour necrosis factor-related apoptosis inducing ligand-mediated apoptosis in the human hepatocellular carcinoma cell line QGY-7703 Eur J Biochem 270: 2721-2731 133 Huang, S, and Sinicrope, FA (2008) BH3 mimetic ABT-737 potentiates TRAIL-mediated apoptotic signaling by unsequestering Bim and Bak in human pancreatic cancer cells Cancer Res 68: 2944-2951 134 Song, JH, Kandasamy, K, and Kraft, AS (2008) ABT-737 induces expression of the death receptor and sensitizes human cancer cells to TRAIL-induced apoptosis J Biol Chem 283: 25003-25013 135 Mott, JL, Bronk, SF, Mesa, RA, Kaufmann, SH, and Gores, GJ (2008) BH3only protein mimetic obatoclax sensitizes cholangiocarcinoma cells to Apo2L/TRAIL-induced apoptosis Mol Cancer Ther 7: 2339-2347 105 136 Huang, S, Okumura, K, and Sinicrope, FA (2009) BH3 mimetic obatoclax enhances TRAIL-mediated apoptosis in human pancreatic cancer cells Clin Cancer Res 15: 150-159 137 Romagnoli, M, Desplanques, G, Maiga, S, Legouill, S, Dreano, M, Bataille, R, and Barille-Nion, S (2007) Canonical nuclear factor kappaB pathway inhibition blocks myeloma cell growth and induces apoptosis in strong synergy with TRAIL Clin Cancer Res 13: 6010-6018 138 Jennewein, C, Karl, S, Baumann, B, Micheau, O, Debatin, KM, and Fulda, S (2012) Identification of a novel pro-apoptotic role of NF-kappaB in the regulation of TRAIL- and CD95-mediated apoptosis of glioblastoma cells Oncogene 31: 1468-1474 139 Khanbolooki, S, Nawrocki, ST, Arumugam, T, Andtbacka, R, Pino, MS, Kurzrock, R, Logsdon, CD, Abbruzzese, JL, and McConkey, DJ (2006) Nuclear factor-kappaB maintains TRAIL resistance in human pancreatic cancer cells Mol Cancer Ther 5: 2251-2260 140 Aydin, C, Sanlioglu, AD, Bisgin, A, Yoldas, B, Dertsiz, L, Karacay, B, Griffith, TS, and Sanlioglu, S (2010) NF-kappaB targeting by way of IKK inhibition sensitizes lung cancer cells to adenovirus delivery of TRAIL BMC Cancer 10: 584 141 Ehrhardt, H, Fulda, S, Schmid, I, Hiscott, J, Debatin, KM, and Jeremias, I (2003) TRAIL induced survival and proliferation in cancer cells resistant towards TRAIL-induced apoptosis mediated by NF-kappaB Oncogene 22: 3842-3852 142 Shrader, M, Pino, MS, Lashinger, L, Bar-Eli, M, Adam, L, Dinney, CP, and McConkey, DJ (2007) Gefitinib reverses TRAIL resistance in human bladder cancer cell lines via inhibition of AKT-mediated X-linked inhibitor of apoptosis protein expression Cancer Res 67: 1430-1435 143 Secchiero, P, Gonelli, A, Carnevale, E, Milani, D, Pandolfi, A, Zella, D, and Zauli, G (2003) TRAIL promotes the survival and proliferation of primary human vascular endothelial cells by activating the Akt and ERK pathways Circulation 107: 2250-2256 144 Zauli, G, Sancilio, S, Cataldi, A, Sabatini, N, Bosco, D, and Di Pietro, R (2005) PI-3K/Akt and NF-kappaB/IkappaBalpha pathways are activated in Jurkat T cells in response to TRAIL treatment J Cell Physiol 202: 900-911 145 Tran, SE, Holmstrom, TH, Ahonen, M, Kahari, VM, and Eriksson, JE (2001) MAPK/ERK overrides the apoptotic signaling from Fas, TNF, and TRAIL receptors J Biol Chem 276: 16484-16490 146 Lee, TJ, Lee, JT, Park, JW, and Kwon, TK (2006) Acquired TRAIL resistance in human breast cancer cells are caused by the sustained cFLIP(L) and XIAP protein levels and ERK activation Biochem Biophys Res Commun 351: 1024-1030 147 Son, JK, Varadarajan, S, and Bratton, SB (2010) TRAIL-activated stress kinases suppress apoptosis through transcriptional upregulation of MCL-1 Cell Death Differ 17: 1288-1301 106 148 Wang, C, Chen, T, Zhang, N, Yang, M, Li, B, Lu, X, Cao, X, and Ling, C (2009) Melittin, a major component of bee venom, sensitizes human hepatocellular carcinoma cells to tumor necrosis factor-related apoptosisinducing ligand (TRAIL)-induced apoptosis by activating CaMKII-TAK1JNK/p38 and inhibiting IkappaBalpha kinase-NFkappaB J Biol Chem 284: 3804-3813 149 Chen, LH, Jiang, CC, Kiejda, KA, Wang, YF, Thorne, RF, Zhang, XD, and Hersey, P (2007) Thapsigargin sensitizes human melanoma cells to TRAILinduced apoptosis by up-regulation of TRAIL-R2 through the unfolded protein response Carcinogenesis 28: 2328-2336 150 Sung, B, Ravindran, J, Prasad, S, Pandey, MK, and Aggarwal, BB (2010) Gossypol induces death receptor-5 through activation of the ROS-ERKCHOP pathway and sensitizes colon cancer cells to TRAIL J Biol Chem 285: 35418-35427 151 Siegelin, MD, Gaiser, T, Habel, A, and Siegelin, Y (2009) Daidzein overcomes TRAIL-resistance in malignant glioma cells by modulating the expression of the intrinsic apoptotic inhibitor, bcl-2 Neurosci Lett 454: 223228 152 Shankar, S, Ganapathy, S, Chen, Q, and Srivastava, RK (2008) Curcumin sensitizes TRAIL-resistant xenografts: molecular mechanisms of apoptosis, metastasis and angiogenesis Mol Cancer 7: 16 153 Jane, EP, Premkumar, DR, and Pollack, IF (2011) Bortezomib sensitizes malignant human glioma cells to TRAIL, mediated by inhibition of the NF{kappa}B signaling pathway Mol Cancer Ther 10: 198-208 154 Inoue, T, Shiraki, K, Fuke, H, Yamanaka, Y, Miyashita, K, Yamaguchi, Y, Yamamoto, N, Ito, K, Sugimoto, K, and Nakano, T (2006) Proteasome inhibition sensitizes hepatocellular carcinoma cells to TRAIL by suppressing caspase inhibitors and AKT pathway Anticancer Drugs 17: 261-268 155 Szegezdi, E, Cahill, S, Meyer, M, O'Dwyer, M, and Samali, A (2006) TRAIL sensitisation by arsenic trioxide is caspase-8 dependent and involves modulation of death receptor components and Akt Br J Cancer 94: 398-406 156 Jin, CY, Moon, DO, Lee, JD, Heo, MS, Choi, YH, Lee, CM, Park, YM, and Kim, GY (2007) Sulforaphane sensitizes tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis through downregulation of ERK and Akt in lung adenocarcinoma A549 cells Carcinogenesis 28: 10581066 157 Giaume, C, Leybaert, L, Naus, CC, and Saez, JC (2013) Connexin and pannexin hemichannels in brain glial cells: properties, pharmacology, and roles Front Pharmacol 4: 88 158 Oyamada, M, Oyamada, Y, and Takamatsu, T (2005) Regulation of connexin expression Biochim Biophys Acta 1719: 6-23 107 159 Willecke, K, Eiberger, J, Degen, J, Eckardt, D, Romualdi, A, Guldenagel, M, Deutsch, U, and Sohl, G (2002) Structural and functional diversity of connexin genes in the mouse and human genome Biol Chem 383: 725-737 160 Solan, JL, and Lampe, PD (2009) Connexin43 phosphorylation: structural changes and biological effects Biochem J 419: 261-272 161 Herve, JC, and Derangeon, M (2012) Gap-junction-mediated cell-to-cell communication Cell Tissue Res 352: 21-31 162 Sagar, GD, and Larson, DM (2006) Carbenoxolone inhibits junctional transfer and upregulates Connexin43 expression by a protein kinase Adependent pathway J Cell Biochem 98: 1543-1551 163 Oliveira, R, Christov, C, Guillamo, JS, de Bouard, S, Palfi, S, Venance, L, Tardy, M, and Peschanski, M (2005) Contribution of gap junctional communication between tumor cells and astroglia to the invasion of the brain parenchyma by human glioblastomas BMC Cell Biol 6: 164 Westhoff, MA, Zhou, S, Bachem, MG, Debatin, KM, and Fulda, S (2008) Identification of a novel switch in the dominant forms of cell adhesionmediated drug resistance in glioblastoma cells Oncogene 27: 5169-5181 165 Verselis, VK, and Srinivas, M (2013) Connexin channel modulators and their mechanisms of action Neuropharmacology 166 Davidson, JS, and Baumgarten, IM (1988) Glycyrrhetinic acid derivatives: a novel class of inhibitors of gap-junctional intercellular communication Structure-activity relationships J Pharmacol Exp Ther 246: 1104-1107 167 Jellinck, PH, Monder, C, McEwen, BS, and Sakai, RR (1993) Differential inhibition of 11 beta-hydroxysteroid dehydrogenase by carbenoxolone in rat brain regions and peripheral tissues J Steroid Biochem Mol Biol 46: 209-213 168 Chin, YW, Balunas, MJ, Chai, HB, and Kinghorn, AD (2006) Drug discovery from natural sources Aaps J 8: E239-253 169 Picoli, C, Nouvel, V, Aubry, F, Reboul, M, Duchene, A, Jeanson, T, Thomasson, J, Mouthon, F, and Charveriat, M (2012) Human connexin channel specificity of classical and new gap junction inhibitors J Biomol Screen 17: 1339-1347 170 Sohl, G, and Willecke, K (2004) Gap junctions and the connexin protein family Cardiovasc Res 62: 228-232 171 Laird, DW (2006) Life cycle of connexins in health and disease Biochem J 394: 527-543 172 Goodenough, DA, and Paul, DL (2003) Beyond the gap: functions of unpaired connexon channels Nat Rev Mol Cell Biol 4: 285-294 173 Laird, DW, Castillo, M, and Kasprzak, L (1995) Gap junction turnover, intracellular trafficking, and phosphorylation of connexin43 in brefeldin Atreated rat mammary tumor cells J Cell Biol 131: 1193-1203 108 174 Lampe, PD (1994) Analyzing phorbol ester effects on gap junctional communication: a dramatic inhibition of assembly J Cell Biol 127: 18951905 175 Lampe, PD, TenBroek, EM, Burt, JM, Kurata, WE, Johnson, RG, and Lau, AF (2000) Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication J Cell Biol 149: 1503-1512 176 Ek-Vitorin, JF, King, TJ, Heyman, NS, Lampe, PD, and Burt, JM (2006) Selectivity of connexin 43 channels is regulated through protein kinase Cdependent phosphorylation Circ Res 98: 1498-1505 177 Dbouk, HA, Mroue, RM, El-Sabban, ME, and Talhouk, RS (2009) Connexins: a myriad of functions extending beyond assembly of gap junction channels Cell Commun Signal 7: 178 Herve, JC, Derangeon, M, Sarrouilhe, D, Giepmans, BN, and Bourmeyster, N (2012) Gap junctional channels are parts of multiprotein complexes Biochim Biophys Acta 1818: 1844-1865 179 Gellhaus, A, Dong, X, Propson, S, Maass, K, Klein-Hitpass, L, Kibschull, M, Traub, O, Willecke, K, Perbal, B, Lye, SJ, and Winterhager, E (2004) Connexin43 interacts with NOV: a possible mechanism for negative regulation of cell growth in choriocarcinoma cells J Biol Chem 279: 3693136942 180 Pu, P, Xia, Z, Yu, S, and Huang, Q (2004) Altered expression of Cx43 in astrocytic tumors Clin Neurol Neurosurg 107: 49-54 181 Soroceanu, L, Manning, TJ, Jr., and Sontheimer, H (2001) Reduced expression of connexin-43 and functional gap junction coupling in human gliomas Glia 33: 107-117 182 Sin, WC, Crespin, S, and Mesnil, M (2012) Opposing roles of connexin43 in glioma progression Biochim Biophys Acta 1818: 2058-2067 183 Zhang, YW, Nakayama, K, Nakayama, K, and Morita, I (2003) A novel route for connexin 43 to inhibit cell proliferation: negative regulation of Sphase kinase-associated protein (Skp 2) Cancer Res 63: 1623-1630 184 Zhang, YW, Morita, I, Ikeda, M, Ma, KW, and Murota, S (2001) Connexin43 suppresses proliferation of osteosarcoma U2OS cells through post-transcriptional regulation of p27 Oncogene 20: 4138-4149 185 Tabernero, A, Sanchez-Alvarez, R, and Medina, JM (2006) Increased levels of cyclins D1 and D3 after inhibition of gap junctional communication in astrocytes J Neurochem 96: 973-982 186 Xu, X, Francis, R, Wei, CJ, Linask, KL, and Lo, CW (2006) Connexin 43mediated modulation of polarized cell movement and the directional migration of cardiac neural crest cells Development 133: 3629-3639 187 Baklaushev, VP, Yusubalieva, GM, Tsitrin, EB, Gurina, OI, Grinenko, NP, Victorov, IV, and Chekhonin, VP (2011) Visualization of Connexin 43- 109 positive cells of glioma and the periglioma zone by means of intravenously injected monoclonal antibodies Drug Deliv 18: 331-337 188 Frisch, SM, and Francis, H (1994) Disruption of epithelial cell-matrix interactions induces apoptosis J Cell Biol 124: 619-626 189 Aoudjit, F, and Vuori, K (2001) Integrin signaling inhibits paclitaxelinduced apoptosis in breast cancer cells Oncogene 20: 4995-5004 190 Hazlehurst, LA, and Dalton, WS (2001) Mechanisms associated with cell adhesion mediated drug resistance (CAM-DR) in hematopoietic malignancies Cancer Metastasis Rev 20: 43-50 191 Cordes, N, Hansmeier, B, Beinke, C, Meineke, V, and van Beuningen, D (2003) Irradiation differentially affects substratum-dependent survival, adhesion, and invasion of glioblastoma cell lines Br J Cancer 89: 2122-2132 192 Nagasawa, K, Chiba, H, Fujita, H, Kojima, T, Saito, T, Endo, T, and Sawada, N (2006) Possible involvement of gap junctions in the barrier function of tight junctions of brain and lung endothelial cells J Cell Physiol 208: 123132 193 Wu, X, and Hui, KM (2004) Induction of potent TRAIL-mediated tumoricidal activity by hFLEX/Furin/TRAIL recombinant DNA construct Mol Ther 9: 674-681 194 Fraefel, C, Song, S, Lim, F, Lang, P, Yu, L, Wang, Y, Wild, P, and Geller, AI (1996) Helper virus-free transfer of herpes simplex virus type plasmid vectors into neural cells J Virol 70: 7190-7197 195 Wang, S (2008) The promise of cancer therapeutics targeting the TNFrelated apoptosis-inducing ligand and TRAIL receptor pathway Oncogene 27: 6207-6215 196 Vidya Priyadarsini, R, Senthil Murugan, R, Maitreyi, S, Ramalingam, K, Karunagaran, D, and Nagini, S (2010) The flavonoid quercetin induces cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells through p53 induction and NF-kappaB inhibition Eur J Pharmacol 649: 84-91 197 Hsieh, TC, and Wu, JM (1999) Differential effects on growth, cell cycle arrest, and induction of apoptosis by resveratrol in human prostate cancer cell lines Exp Cell Res 249: 109-115 198 Weir, NM, Selvendiran, K, Kutala, VK, Tong, L, Vishwanath, S, Rajaram, M, Tridandapani, S, Anant, S, and Kuppusamy, P (2007) Curcumin induces G2/M arrest and apoptosis in cisplatin-resistant human ovarian cancer cells by modulating Akt and p38 MAPK Cancer Biol Ther 6: 178-184 199 Ling, YH, Liebes, L, Jiang, JD, Holland, JF, Elliott, PJ, Adams, J, Muggia, FM, and Perez-Soler, R (2003) Mechanisms of proteasome inhibitor PS-341induced G(2)-M-phase arrest and apoptosis in human non-small cell lung cancer cell lines Clin Cancer Res 9: 1145-1154 110 200 Pivato, LS, Constantin, RP, Ishii-Iwamoto, EL, Kelmer-Bracht, AM, Yamamoto, NS, Constantin, J, and Bracht, A (2006) Metabolic effects of carbenoxolone in rat liver J Biochem Mol Toxicol 20: 230-240 201 Fletcher, MJ, MacKay, N, and Forbes, CD (1967) Assessment of possible glucocorticoid activity of carbenoxolone sodium Br Med J 1: 412-413 202 Du, J, Wang, Y, Hunter, R, Wei, Y, Blumenthal, R, Falke, C, Khairova, R, Zhou, R, Yuan, P, Machado-Vieira, R, McEwen, BS, and Manji, HK (2009) Dynamic regulation of mitochondrial function by glucocorticoids Proc Natl Acad Sci U S A 106: 3543-3548 203 Leshchenko, Y, Likhodii, S, Yue, W, Burnham, WM, and Perez Velazquez, JL (2006) Carbenoxolone does not cross the blood brain barrier: an HPLC study BMC Neurosci 7: 204 Zhuang, S, Demirs, JT, and Kochevar, IE (2000) p38 mitogen-activated protein kinase mediates bid cleavage, mitochondrial dysfunction, and caspase-3 activation during apoptosis induced by singlet oxygen but not by hydrogen peroxide J Biol Chem 275: 25939-25948 205 Herrero-Gonzalez, S, Valle-Casuso, JC, Sanchez-Alvarez, R, Giaume, C, Medina, JM, and Tabernero, A (2009) Connexin43 is involved in the effect of endothelin-1 on astrocyte proliferation and glucose uptake Glia 57: 222233 206 Lampe, PD, and Lau, AF (2004) The effects of connexin phosphorylation on gap junctional communication Int J Biochem Cell Biol 36: 1171-1186 207 Bellail, AC, Qi, L, Mulligan, P, Chhabra, V, and Hao, C (2009) TRAIL agonists on clinical trials for cancer therapy: the promises and the challenges Rev Recent Clin Trials 4: 34-41 208 Herbst, RS, Eckhardt, SG, Kurzrock, R, Ebbinghaus, S, O'Dwyer, PJ, Gordon, MS, Novotny, W, Goldwasser, MA, Tohnya, TM, Lum, BL, Ashkenazi, A, Jubb, AM, and Mendelson, DS (2010) Phase I dose-escalation study of recombinant human Apo2L/TRAIL, a dual proapoptotic receptor agonist, in patients with advanced cancer J Clin Oncol 28: 2839-2846 209 Bellail, AC, and Hao, C (2012) The roadmap of TRAIL apoptotic pathwaytargeted cancer therapies: What is next? Expert Rev Anticancer Ther 12: 547549 210 Thorburn, A, Behbakht, K, and Ford, H (2008) TRAIL receptor-targeted therapeutics: resistance mechanisms and strategies to avoid them Drug Resist Updat 11: 17-24 211 von Pawel, J, Harvey, JH, Spigel, DR, Dediu, M, Reck, M, Cebotaru, CL, Humphreys, RC, Gribbin, MJ, Fox, NL, and Camidge, DR (2014) Phase II Trial of Mapatumumab, a Fully Human Agonist Monoclonal Antibody to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor (TRAIL-R1), in Combination With Paclitaxel and Carboplatin in Patients With Advanced Non-Small-Cell Lung Cancer Clin Lung Cancer 15: 188-196 e182 111 212 Reck, M, Krzakowski, M, Chmielowska, E, Sebastian, M, Hadler, D, Fox, T, Wang, Q, Greenberg, J, Beckman, RA, and von Pawel, J (2013) A randomized, double-blind, placebo-controlled phase study of tigatuzumab (CS-1008) in combination with carboplatin/paclitaxel in patients with chemotherapy-naive metastatic/unresectable non-small cell lung cancer Lung Cancer 82: 441-448 213 Loebinger, MR, Eddaoudi, A, Davies, D, and Janes, SM (2009) Mesenchymal stem cell delivery of TRAIL can eliminate metastatic cancer Cancer Res 69: 4134-4142 214 Kim, SM, Lim, JY, Park, SI, Jeong, CH, Oh, JH, Jeong, M, Oh, W, Park, SH, Sung, YC, and Jeun, SS (2008) Gene therapy using TRAIL-secreting human umbilical cord blood-derived mesenchymal stem cells against intracranial glioma Cancer Res 68: 9614-9623 215 Menon, LG, Kelly, K, Wei Yang, H, Kim, SK, Black, PM, and Carroll, RS (2009) Human Bone Marrow Derived Mesenchymal Stromal Cells Expressing S-TRAIL as a Cellular Delivery Vehicle for Human Glioma Therapy Stem Cells 216 Wiley, SR, Schooley, K, Smolak, PJ, Din, WS, Huang, CP, Nicholl, JK, Sutherland, GR, Smith, TD, Rauch, C, Smith, CA, and et al (1995) Identification and characterization of a new member of the TNF family that induces apoptosis Immunity 3: 673-682 217 Walczak, H, Miller, RE, Ariail, K, Gliniak, B, Griffith, TS, Kubin, M, Chin, W, Jones, J, Woodward, A, Le, T, Smith, C, Smolak, P, Goodwin, RG, Rauch, CT, Schuh, JC, and Lynch, DH (1999) Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo Nat Med 5: 157-163 218 Kim, SH, Kim, K, Kwagh, JG, Dicker, DT, Herlyn, M, Rustgi, AK, Chen, Y, and El-Deiry, WS (2004) Death induction by recombinant native TRAIL and its prevention by a caspase inhibitor in primary human esophageal epithelial cells J Biol Chem 279: 40044-40052 219 van der Sloot, AM, Tur, V, Szegezdi, E, Mullally, MM, Cool, RH, Samali, A, Serrano, L, and Quax, WJ (2006) Designed tumor necrosis factor-related apoptosis-inducing ligand variants initiating apoptosis exclusively via the DR5 receptor Proc Natl Acad Sci U S A 103: 8634-8639 220 Tur, V, van der Sloot, AM, Reis, CR, Szegezdi, E, Cool, RH, Samali, A, Serrano, L, and Quax, WJ (2008) DR4-selective tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) variants obtained by structurebased design J Biol Chem 283: 20560-20568 221 Valley, CC, Cembran, A, Perlmutter, JD, Lewis, AK, Labello, NP, Gao, J, and Sachs, JN (2012) The methionine-aromatic motif plays a unique role in stabilizing protein structure J Biol Chem 287: 34979-34991 222 Timmers, L, Lim, SK, Arslan, F, Armstrong, JS, Hoefer, IE, Doevendans, PA, Piek, JJ, El Oakley, RM, Choo, A, Lee, CN, Pasterkamp, G, and de Kleijn, DP (2007) Reduction of myocardial infarct size by human mesenchymal stem cell conditioned medium Stem Cell Res 1: 129-137 112 223 Yew, TL, Hung, YT, Li, HY, Chen, HW, Chen, LL, Tsai, KS, Chiou, SH, Chao, KC, Huang, TF, Chen, HL, and Hung, SC (2011) Enhancement of wound healing by human multipotent stromal cell conditioned medium: the paracrine factors and p38 MAPK activation Cell Transplant 20: 693-706 224 Park, KS, Kim, YS, Kim, JH, Choi, B, Kim, SH, Tan, AH, Lee, MS, Lee, MK, Kwon, CH, Joh, JW, Kim, SJ, and Kim, KW (2010) Trophic molecules derived from human mesenchymal stem cells enhance survival, function, and angiogenesis of isolated islets after transplantation Transplantation 89: 509517 225 Cantinieaux, D, Quertainmont, R, Blacher, S, Rossi, L, Wanet, T, Noel, A, Brook, G, Schoenen, J, and Franzen, R (2013) Conditioned medium from bone marrow-derived mesenchymal stem cells improves recovery after spinal cord injury in rats: an original strategy to avoid cell transplantation PLoS One 8: e69515 226 Gauthaman, K, Yee, FC, Cheyyatraivendran, S, Biswas, A, Choolani, M, and Bongso, A (2012) Human umbilical cord Wharton's jelly stem cell (hWJSC) extracts inhibit cancer cell growth in vitro J Cell Biochem 113: 2027-2039 227 Bruno, S, Collino, F, Deregibus, MC, Grange, C, Tetta, C, and Camussi, G (2013) Microvesicles derived from human bone marrow mesenchymal stem cells inhibit tumor growth Stem Cells Dev 22: 758-771 228 Ho, IA, Ng, WH, and Lam, PY (2010) FasL and FADD delivery by a glioma-specific and cell cycle-dependent HSV-1 amplicon virus enhanced apoptosis in primary human brain tumors Mol Cancer 9: 270 229 Cotrina, ML, Lin, JH, and Nedergaard, M (2008) Adhesive properties of connexin hemichannels Glia 56: 1791-1798 230 Ho, IA, Chan, KY, Ng, WH, Guo, CM, Hui, KM, Cheang, P, and Lam, PY (2009) Matrix metalloproteinase is necessary for the migration of human bone marrow-derived mesenchymal stem cells toward human glioma Stem Cells 27: 1366-1375 231 Sia, KC, Huynh, H, Chinnasamy, N, Hui, KM, and Lam, PY (2012) Suicidal gene therapy in the effective control of primary human hepatocellular carcinoma as monitored by noninvasive bioimaging Gene Ther 19: 532-542 232 Sia, KC, Chong, WK, Ho, IA, Yulyana, Y, Endaya, B, Huynh, H, and Lam, PY (2010) Hybrid herpes simplex virus/Epstein-Barr virus amplicon viral vectors confer enhanced transgene expression in primary human tumors and human bone marrow-derived mesenchymal stem cells J Gene Med 12: 848858 233 Kelley, SK, Harris, LA, Xie, D, Deforge, L, Totpal, K, Bussiere, J, and Fox, JA (2001) Preclinical studies to predict the disposition of Apo2L/tumor necrosis factor-related apoptosis-inducing ligand in humans: characterization of in vivo efficacy, pharmacokinetics, and safety J Pharmacol Exp Ther 299: 31-38 234 Rozanov, DV, Savinov, AY, Golubkov, VS, Rozanova, OL, Postnova, TI, Sergienko, EA, Vasile, S, Aleshin, AE, Rega, MF, Pellecchia, M, and 113 Strongin, AY (2009) Engineering a leucine zipper-TRAIL homotrimer with improved cytotoxicity in tumor cells Mol Cancer Ther 8: 1515-1525 235 Winkeler, A, Sena-Esteves, M, Paulis, LE, Li, H, Waerzeggers, Y, Ruckriem, B, Himmelreich, U, Klein, M, Monfared, P, Rueger, MA, Heneka, M, Vollmar, S, Hoehn, M, Fraefel, C, Graf, R, Wienhard, K, Heiss, WD, and Jacobs, AH (2007) Switching on the lights for gene therapy PLoS One 2: e528 236 Lee, JY, Lee, DH, Kim, HA, Choi, SA, Lee, HJ, Park, CK, Phi, JH, Wang, KC, Kim, SU, and Kim, SK (2013) Double suicide gene therapy using human neural stem cells against glioblastoma: double safety measures J Neurooncol 237 Kim, SW, Kim, SJ, Park, SH, Yang, HG, Kang, MC, Choi, YW, Kim, SM, Jeun, SS, and Sung, YC (2013) Complete regression of metastatic renal cell carcinoma by multiple injections of engineered mesenchymal stem cells expressing dodecameric TRAIL and HSV-TK Clin Cancer Res 19: 415-427 238 Plotkin, LI, Manolagas, SC, and Bellido, T (2002) Transduction of cell survival signals by connexin-43 hemichannels J Biol Chem 277: 8648-8657 239 Mesnil, M, Crespin, S, Avanzo, JL, and Zaidan-Dagli, ML (2005) Defective gap junctional intercellular communication in the carcinogenic process Biochim Biophys Acta 1719: 125-145 240 Cottin, S, Gould, PV, Cantin, L, and Caruso, M (2011) Gap junctions in human glioblastomas: implications for suicide gene therapy Cancer Gene Ther 18: 674-681 241 Cottin, S, Ghani, K, and Caruso, M (2008) Bystander effect in glioblastoma cells with a predominant cytoplasmic localization of connexin43 Cancer Gene Ther 15: 823-831 242 Boengler, K, Dodoni, G, Rodriguez-Sinovas, A, Cabestrero, A, Ruiz-Meana, M, Gres, P, Konietzka, I, Lopez-Iglesias, C, Garcia-Dorado, D, Di Lisa, F, Heusch, G, and Schulz, R (2005) Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning Cardiovasc Res 67: 234-244 243 Rodriguez-Sinovas, A, Boengler, K, Cabestrero, A, Gres, P, Morente, M, Ruiz-Meana, M, Konietzka, I, Miro, E, Totzeck, A, Heusch, G, Schulz, R, and Garcia-Dorado, D (2006) Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection Circ Res 99: 93-101 244 Ruiz-Meana, M, Rodriguez-Sinovas, A, Cabestrero, A, Boengler, K, Heusch, G, and Garcia-Dorado, D (2008) Mitochondrial connexin43 as a new player in the pathophysiology of myocardial ischaemia-reperfusion injury Cardiovasc Res 77: 325-333 245 Jensen, K, Patel, A, Klubo-Gwiezdzinska, J, Bauer, A, and Vasko, V (2011) Inhibition of gap junction transfer sensitizes thyroid cancer cells to anoikis Endocr Relat Cancer 18: 613-626 114 246 Sharrow, AC, Li, Y, Micsenyi, A, Griswold, RD, Wells, A, Monga, SS, and Blair, HC (2008) Modulation of osteoblast gap junction connectivity by serum, TNFalpha, and TRAIL Exp Cell Res 314: 297-308 247 Hao, JL, Suzuki, K, Lu, Y, Hirano, S, Fukuda, K, Kumagai, N, Kimura, K, and Nishida, T (2005) Inhibition of gap junction-mediated intercellular communication by TNF-alpha in cultured human corneal fibroblasts Invest Ophthalmol Vis Sci 46: 1195-1200 248 Theiss, C, Mazur, A, Meller, K, and Mannherz, HG (2007) Changes in gap junction organization and decreased coupling during induced apoptosis in lens epithelial and NIH-3T3 cells Exp Cell Res 313: 38-52 249 Olbina, G, and Eckhart, W (2003) Mutations in the second extracellular region of connexin 43 prevent localization to the plasma membrane, but not affect its ability to suppress cell growth Mol Cancer Res 1: 690-700 250 Herrero-Gonzalez, S, Gangoso, E, Giaume, C, Naus, CC, Medina, JM, and Tabernero, A (2010) Connexin43 inhibits the oncogenic activity of c-Src in C6 glioma cells Oncogene 29: 5712-5723 251 Gielen, PR, Aftab, Q, Ma, N, Chen, VC, Hong, X, Lozinsky, S, Naus, CC, and Sin, WC (2013) Connexin43 confers Temozolomide resistance in human glioma cells by modulating the mitochondrial apoptosis pathway Neuropharmacology 252 Zundorf, G, Kahlert, S, and Reiser, G (2007) Gap-junction blocker carbenoxolone differentially enhances NMDA-induced cell death in hippocampal neurons and astrocytes in co-culture J Neurochem 102: 508521 253 Salvi, M, Fiore, C, Battaglia, V, Palermo, M, Armanini, D, and Toninello, A (2005) Carbenoxolone induces oxidative stress in liver mitochondria, which is responsible for transition pore opening Endocrinology 146: 2306-2312 254 Azarashvili, T, Baburina, Y, Grachev, D, Krestinina, O, Evtodienko, Y, Stricker, R, and Reiser, G (2011) Calcium-induced permeability transition in rat brain mitochondria is promoted by carbenoxolone through targeting connexin43 Am J Physiol Cell Physiol 300: C707-720 255 Lee, HJ, Lee, HJ, Sohn, EJ, Lee, EO, Kim, JH, Lee, MH, and Kim, SH (2012) Inhibition of Connexin 26/43 and Extracellular-Regulated Kinase Protein Plays a Critical Role in Melatonin Facilitated Gap Junctional Intercellular Communication in Hydrogen Peroxide-Treated HaCaT Keratinocyte Cells Evid Based Complement Alternat Med 2012: 589365 256 Woo, JS, Kim, SM, Jeong, CH, Ryu, CH, and Jeun, SS (2013) Lipoxygenase inhibitor MK886 potentiates TRAIL-induced apoptosis through CHOP- and p38 MAPK-mediated up-regulation of death receptor in malignant glioma Biochem Biophys Res Commun 431: 354-359 257 Nishitoh, H (2012) CHOP is a multifunctional transcription factor in the ER stress response J Biochem 151: 217-219 115 258 McCullough, KD, Martindale, JL, Klotz, LO, Aw, TY, and Holbrook, NJ (2001) Gadd153 sensitizes cells to endoplasmic reticulum stress by downregulating Bcl2 and perturbing the cellular redox state Mol Cell Biol 21: 1249-1259 259 Yamaguchi, H, and Wang, HG (2004) CHOP is involved in endoplasmic reticulum stress-induced apoptosis by enhancing DR5 expression in human carcinoma cells J Biol Chem 279: 45495-45502 260 Jung, EM, Park, JW, Choi, KS, Park, JW, Lee, HI, Lee, KS, and Kwon, TK (2006) Curcumin sensitizes tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis through CHOP-independent DR5 upregulation Carcinogenesis 27: 2008-2017 261 Yoshida, T, Shiraishi, T, Nakata, S, Horinaka, M, Wakada, M, Mizutani, Y, Miki, T, and Sakai, T (2005) Proteasome inhibitor MG132 induces death receptor through CCAAT/enhancer-binding protein homologous protein Cancer Res 65: 5662-5667 262 Wu, GS, Burns, TF, McDonald, ER, 3rd, Meng, RD, Kao, G, Muschel, R, Yen, T, and el-Deiry, WS (1999) Induction of the TRAIL receptor KILLER/DR5 in p53-dependent apoptosis but not growth arrest Oncogene 18: 6411-6418 263 Prasad, S, Yadav, VR, Kannappan, R, and Aggarwal, BB (2011) Ursolic acid, a pentacyclin triterpene, potentiates TRAIL-induced apoptosis through p53independent up-regulation of death receptors: evidence for the role of reactive oxygen species and JNK J Biol Chem 286: 5546-5557 264 Gupta, SC, Francis, SK, Nair, MS, Mo, YY, and Aggarwal, BB (2013) Azadirone, a Limonoid Tetranortriterpene, Induces Death Receptors and Sensitizes Human Cancer Cells to Tumor Necrosis Factor-related Apoptosisinducing Ligand (TRAIL) through a p53 Protein-independent Mechanism: EVIDENCE FOR THE ROLE OF THE ROS-ERK-CHOP-DEATH RECEPTOR PATHWAY J Biol Chem 288: 32343-32356 265 Parke, DV (1983) The biochemical pharmacology of carbenoxolone Its possible mechanisms of action Acta Gastroenterol Belg 46: 437-447 266 Takeuchi, H, Mizoguchi, H, Doi, Y, Jin, S, Noda, M, Liang, J, Li, H, Zhou, Y, Mori, R, Yasuoka, S, Li, E, Parajuli, B, Kawanokuchi, J, Sonobe, Y, Sato, J, Yamanaka, K, Sobue, G, Mizuno, T, and Suzumura, A (2011) Blockade of gap junction hemichannel suppresses disease progression in mouse models of amyotrophic lateral sclerosis and Alzheimer's disease PLoS One 6: e21108 267 Zauli, G, Milani, D, Rimondi, E, Baldini, G, Nicolin, V, Grill, V, and Secchiero, P (2003) TRAIL activates a caspase 9/7-dependent pathway in caspase 8/10-defective SK-N-SH neuroblastoma cells with two functional end points: induction of apoptosis and PGE2 release Neoplasia 5: 457-466 268 Sato, T, Machida, T, Takahashi, S, Iyama, S, Sato, Y, Kuribayashi, K, Takada, K, Oku, T, Kawano, Y, Okamoto, T, Takimoto, R, Matsunaga, T, Takayama, T, Takahashi, M, Kato, J, and Niitsu, Y (2004) Fas-mediated apoptosome formation is dependent on reactive oxygen species derived from mitochondrial permeability transition in Jurkat cells J Immunol 173: 285-296 116 269 Katoh, I, Tomimori, Y, Ikawa, Y, and Kurata, S (2004) Dimerization and processing of procaspase-9 by redox stress in mitochondria J Biol Chem 279: 15515-15523 270 Wang, X (2001) The expanding role of mitochondria in apoptosis Genes Dev 15: 2922-2933 271 Perlman, H, Zhang, X, Chen, MW, Walsh, K, and Buttyan, R (1999) An elevated bax/bcl-2 ratio corresponds with the onset of prostate epithelial cell apoptosis Cell Death Differ 6: 48-54 272 Raisova, M, Hossini, AM, Eberle, J, Riebeling, C, Wieder, T, Sturm, I, Daniel, PT, Orfanos, CE, and Geilen, CC (2001) The Bax/Bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fasmediated apoptosis J Invest Dermatol 117: 333-340 273 Hockenbery, DM, Oltvai, ZN, Yin, XM, Milliman, CL, and Korsmeyer, SJ (1993) Bcl-2 functions in an antioxidant pathway to prevent apoptosis Cell 75: 241-251 274 Pugazhenthi, S, Nesterova, A, Jambal, P, Audesirk, G, Kern, M, Cabell, L, Eves, E, Rosner, MR, Boxer, LM, and Reusch, JE (2003) Oxidative stressmediated down-regulation of bcl-2 promoter in hippocampal neurons J Neurochem 84: 982-996 275 Li, D, Ueta, E, Kimura, T, Yamamoto, T, and Osaki, T (2004) Reactive oxygen species (ROS) control the expression of Bcl-2 family proteins by regulating their phosphorylation and ubiquitination Cancer Sci 95: 644-650 276 Lee, MW, Park, SC, Yang, YG, Yim, SO, Chae, HS, Bach, JH, Lee, HJ, Kim, KY, Lee, WB, and Kim, SS (2002) The involvement of reactive oxygen species (ROS) and p38 mitogen-activated protein (MAP) kinase in TRAIL/Apo2L-induced apoptosis FEBS Lett 512: 313-318 277 Sung, B, Prasad, S, Ravindran, J, Yadav, VR, and Aggarwal, BB (2012) Capsazepine, a TRPV1 antagonist, sensitizes colorectal cancer cells to apoptosis by TRAIL through ROS-JNK-CHOP-mediated upregulation of death receptors Free Radic Biol Med 53: 1977-1987 278 Kim, SM, Woo, JS, Jeong, CH, Ryu, CH, Jang, JD, and Jeun, SS (2014) Potential application of temozolomide in mesenchymal stem cell-based TRAIL gene therapy against malignant glioma Stem Cells Transl Med 3: 172-182 279 Munoz, JL, Rodriguez-Cruz, V, Greco, SJ, Ramkissoon, SH, Ligon, KL, and Rameshwar, P (2014) Temozolomide resistance in glioblastoma cells occurs partly through epidermal growth factor receptor-mediated induction of connexin 43 Cell Death Dis 5: e1145 280 Kim, SM, Oh, JH, Park, SA, Ryu, CH, Lim, JY, Kim, DS, Chang, JW, Oh, W, and Jeun, SS (2010) Irradiation enhances the tumor tropism and therapeutic potential of tumor necrosis factor-related apoptosis-inducing ligand-secreting human umbilical cord blood-derived mesenchymal stem cells in glioma therapy Stem Cells 28: 2217-2228 117 281 Stoletov, K, Strnadel, J, Zardouzian, E, Momiyama, M, Park, FD, Kelber, JA, Pizzo, DP, Hoffman, R, VandenBerg, SR, and Klemke, RL (2013) Role of connexins in metastatic breast cancer and melanoma brain colonization J Cell Sci 126: 904-913 282 Mandel, K, Yang, Y, Schambach, A, Glage, S, Otte, A, and Hass, R (2013) Mesenchymal stem cells directly interact with breast cancer cells and promote tumor cell growth in vitro and in vivo Stem Cells Dev 22: 31143127 283 Roorda, BD, Elst, A, Boer, TG, Kamps, WA, and de Bont, ES (2010) Mesenchymal stem cells contribute to tumor cell proliferation by direct cellcell contact interactions Cancer Invest 28: 526-534 284 Lis, R, Touboul, C, Raynaud, CM, Malek, JA, Suhre, K, Mirshahi, M, and Rafii, A (2012) Mesenchymal cell interaction with ovarian cancer cells triggers pro-metastatic properties PLoS One 7: e38340 285 Zhang, X, Sun, Y, Wang, Z, Huang, Z, Li, B, and Fu, J (2014) Upregulation of Cx43 Expression in Bone Marrow Mesenchymal Stem Cells Plays a Crucial Role in Adhesion and Migration of Multiple Myeloma Cells Leuk Lymphoma 118 APPENDIX – List of publications Yulyana, Y, Endaya, BB, Ng, WH, Guo, CM, Hui, KM, Lam, PY, et al (2013) Carbenoxolone enhances TRAIL-induced apoptosis through the upregulation of death receptor and inhibition of gap junction intercellular communication in human glioma Stem Cells Dev 22(13):1870-82 Sia, KC, Chong, WK, Ho, IAW, Yulyana, Y, Endaya B, et al (2010) Hybrid herpes simplex virus/Epstein-Barr virus amplicon viral vectors confer enhanced transgene expression in primary human tumors and human bone marrow-derived mesenchymal stem cells J Gene Med 12(10): 848-58 119 ... blue-stained cells over total cells (blue-stained cells + unstained cells) multiplied by 100 2.9 Caspase activity assay Caspase activity was determined using ApoAlert Caspase Colorimetric Assay Kit... Expression level of caspase also plays a role in determining the sensitivity to TRAIL Increase in caspase degradation, as well as silencing of caspase through methylation, has been reported to... DISC, caspase is activated through dimerization and cleavage Activated caspase triggers the activation of the downstream effector caspase 3, which leads to subsequent cleavage of caspase substrates