Induction of Mitochondrial Pathways and Endoplasmic Reticulum Stress for Increasing Apoptosis in Ectopic and Orthotopic Neuroblastoma Xenografts — Oak Academic Publishing
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Induction of Mitochondrial Pathways and Endoplasmic Reticulum Stress for Increasing Apoptosis in Ectopic and Orthotopic Neuroblastoma Xenografts
Cancers are characterized by deregulation of multiple signaling pathways and thus monotherapies are hardly effective. Neuroblastoma, which often occurs in adrenal glands, is the most common childhood malignancy. Malignant neuroblastoma resists traditional treatments and further studies are needed for effective therapeutic interventions. We evaluated synergistic efficacy of N-(4-hydroxyphenyl) retinamide (4-HPR) and genistein (GST) for induction of apoptosis in human malignant neuroblastoma SH-SY5Y and SK-N-BE2 cells in culture and activation of multiple pathways for increasing apoptosis in ectopic and orthotopic neuroblastoma xenografts in nude mice. Combination of 4-HPR and GST synergistically reduced cell viability, caused subG1 accumulation, increased caspase-3 activity for apoptosis in vitro and reduced tumor growth in vivo. Western blotting indicated that combination therapy down regulated Id2 to induce differentiation, increased pro-apoptotic Bax and decreased anti-apoptotic Bcl-2 leading to an increase in Bax:Bcl-2 ratio, increased mitochondrial Bax level, caused mitochondrial release of Smac/Diablo, down regulation of the baculovirus inhibitor-of-apoptosis repeat containing (BIRC) proteins such as BIRC-2 and BIRC-3, and activation of calpain and caspase-3 in SH-SY5Y xenografts. Accumulation of apoptosis-inducing-factor (AIF) in cytosol and increase in caspase-4 activation suggested involvement of mitochondrial pathway and endoplasmic reticulum (ER) stress, respectively, for apoptosis in SH-SY5Y xenografts. In situ immunofluorescent labelings of SH-SY5Y and SK-N-BE2 xenograft sections showed overexpression of calpain, caspase-12, and caspase-3, and AIF, suggesting induction of mitochondrial caspase-dependent and caspase-independent pathways for apoptosis. Collectively, synergistic effects of 4-HPR and GST induced mitochondrial pathways and also ER stress for increasing apoptosis in ectopic and orthotopic neuroblastoma xenografts in nude mice.
S. Y. Sun and R. Lotan, “Retinoids and Their Receptors in Cancer Development and Chemoprevention,” Critical Reviews in Oncology Hematology, Vol. 41, No. 1, 2002, pp. 41-55. doi:10.1016/S1040-8428(01)00144-5
A. L. Sabichi, H. Xu, S. Fischer, C. Zou, X. Yang, V. E. Steele, G. J. Kelloff, R. Lotan and J. L. Clifford, “Retinoid Receptor-Dependent and Independent Biological Activities of Novel Fenretinide Analogues and Metabolites,” Clinical Cancer Research, Vol. 9, No. 12, 2003, pp. 4606-4613.
N. Hail Jr., H. J. Kim and R. Lotan, “Mechanisms of Fenretinide-Induced Apoptosis,” Apoptosis, Vol. 11, No. 10, 2006, pp. 1677-1694. doi:10.1007/s10495-006-9289-3
Q. D. Hewson, P. E. Lovat, M. Corazzari, J. B. Catterall and C. P. Redfern, “The NF-κB Pathway Mediates Fenretinide-Induced Apoptosis in SH-SY5Y Neuroblastoma cells,” Apoptosis, Vol. 10, No. 3, 2005, pp. 493-498. doi:10.1007/s10495-005-1878-z
A. Garaventa, R. Luksch, L. M. S. Piccolo, E. Cavadini, P. G. Montaldo, M. R. Pizzitola, L. Boni, M. Ponzoni, A. Decensi, B. De Bernardi, F. F. Bellani and F. Formelli, “Phase I Trial and Pharmacokinetics of Fenretinide in Children with Neuroblastoma,” Clinical Cancer Research, Vol. 9, No. 6, 2003, pp. 2032-2039.
S. Chen, R. N. Fariss, R. K. Kutty, R. Nelson and B. Wiggert, “Fenretinide-Induced Neuronal Differentiation of ARPE-19 Human Retinal Pigment Epithelial Cells is Associated with the Differential Expression of Hsp70, 14-3-3, Pax-6, Tubulin β-III, NSE, and Bag-1 Proteins,” Molecular Vision, Vol. 12, No. 11, 2006, pp. 1355-1363.
E. J. Park and J. M. Pezzuto, “Botanicals in Cancer Chemoprevention,” Cancer and Metastasis Reviews, Vol. 21, No. 3-4, 2002, pp. 231-255. doi:10.1023/A:1021254725842
F. H. Sarkar and Y. Li, “The Role of Isoflavones in Cancer Chemoprevention,” Frontiers in Bioscience, Vol. 9, No. 1, 2004, pp. 2714-2724. doi:10.2741/1430
T. C. Yeh, P. C. Chiang, T. K. Li, J. L. Hsu, C. J. Lin, S. W. Wang, C. Y. Peng and J. H. Guh, “Genistein Induces Apoptosis in Human Hepatocellular Carcinomas via Interaction of Endoplasmic Reticulum Stress and Mitochondrial Insult,” Biochemical Pharmacology, Vol. 73, No. 6, 2007, pp. 782-792. doi:10.1016/j.bcp.2006.11.027
I. N. Sergeev, “Genistein Induces Ca2+-Mediated, Calpain/Caspase-12-Dependent Apoptosis in Breast Cancer Cells,” Biochemical and Biophysical Research Communications, Vol. 321, No. 2, 2004, pp. 462-467. doi:10.1016/j.bbrc.2004.06.173
A. Brown, P. Jolly and H. Wei, “Genistein Modulates Neuroblastoma Cell Proliferation and Differentiation through Induction of Apoptosis and Regulation of Tyrosine Kinase Activity and N-myc Expression,” Carcinogenesis, Vol. 19, No. 6, 1998, pp. 991-997. doi:10.1093/carcin/19.6.991
A. Das, N. L. Banik and S. K. Ray, “Mechanism of Apoptosis with the Involvement of Calpain and Caspase Cascades in Human Malignant Neuroblastoma SH-SY5Y Cells Exposed to Flavonoids,” International Journal of Cancer, Vol. 119, No. 11, 2006, pp. 2575-2585. doi:10.1002/ijc.22228
G. M. Brodeur, “Neuroblastoma: Biological Insights into a Clinical Enigma,” Nature Reviews Cancer, Vol. 3, No. 3, 2003, pp. 203-216. doi:10.1038/nrc1014
S. Irshad, R. B. Pedley, J. Anderson, D. S. Latchman and V. Budhram-Mahadeo, “The Brn-3b Transcription Factor Regulates the Growth, Behavior, and Invasiveness of Human Neuroblastoma Cells in Vitro and in Vivo,” The Journal of Biological Chemistry, Vol. 279, No. 20, 2004, pp. 21617-21627. doi:10.1074/jbc.M312506200
J. M. Maris and K. K. Matthay, “Molecular Biology of Neuroblastoma,” Journal of Clinical Oncology, Vol. 17, No. 7, 1999, pp. 2264-2279.
R. Torkin, J. F. Lavoie, D. R. Kaplan and H. Yeger, “Induction of Caspase-Dependent, p53-Mediated Apoptosis by Apigenin in Human Neuroblastoma,” Molecular Cancer Therapeutics, Vol. 4, No. 1, 2005, pp. 1-11.
S. Karmakar, K. A. Davis, S. R. Choudhury, A. Deeconda, N. L. Banik and S. K. Ray, “Bcl-2 Inhibitor and Apigenin Worked Synergistically in Human Malignant Neuroblastoma Cell Lines and Increased Apoptosis with Activation of Extrinsic and Intrinsic Pathways,” Biochemical and Biophysical Research Communications, Vol. 388, No. 4, 2009, pp. 705-710. doi:10.1016/j.bbrc.2009.08.071
J. M. Joseph, N. Gross, N. Lassau, V. Rouffiac, P. Opolon, L. Laudani, K. Auderset, J. F. Geay, A. Mühlethaler-Mottet and G. Vassal, “In Vivo Echographic Evidence of Tumoral Vascularization and Microenvironment Interactions in Metastatic Orthotopic Human Neuroblastoma Xenografts,” International Journal of Cancer, Vol. 113, No. 6, 2005, pp. 881-890. doi:10.1002/ijc.20681
S. Karmakar, N. L. Banik, S. J. Patel and S. K. Ray, “Combination of All-Trans Retinoic Acid and Taxol Regressed Glioblastoma T98G Xenografts in Nude Mice,” Apoptosis, Vol. 12, No. 11, 2007, pp. 2077-2087. doi:10.1007/s10495-007-0116-2
S. Karmakar, N. L. Banik and S. K. Ray, “Combination of All-Trans Retinoic Acid and Paclitaxel-Induced Differentiation and Apoptosis in Human Glioblastoma U87MG Xenografts in Nude Mice,” Cancer, Vol. 112, No. 3, 2008, pp. 596-607. doi:10.1002/cncr.23223
C. Du, M. Fang, Y. Li, L. Li and X. Wang, “Smac, a Mitochondrial Protein that Promotes Cytochrome c-Dependent Caspase Activation by Eliminating IAP Inhibition,” Cell, Vol. 102, No. 1, 2000, pp. 33-42. doi:10.1016/S0092-8674(00)00008-8
K. Beppu, J. Jaboine, M. S. Merchant, C. L. Mackall and C. J. Thiele, “Effect of Imatinib Mesylate on Neuroblastoma Tumorigenesis and Vascular Endothelial Growth Factor Expression,” Journal of the National Cancer Institute, Vol. 96, No. 1, 2004, pp. 46-55. doi:10.1093/jnci/djh004
J. L. Armstrong, G. J. Veal, C. P. Redfern and P. E. Lovat, “Role of Noxa in p53-Independent Fenretinide-Induced Apoptosis of Neuroectodermal Tumours,” Apoptosis, Vol. 12, No. 3, 2007, pp. 613-622. doi:10.1007/s10495-006-0020-1
M. Gleichmann, G. Buchheim, H. El-Bizri, Y. Yokota, T. Klockgether, S. Kügler, M. B?hr, M. Weller and J. B. Schulz, “Identification of Inhibitor-of-Differentiation 2 (Id2) as a Modulator of Neuronal Apoptosis,” Journal of Neurochemistry, Vol. 80, No. 5, 2002, pp. 755-762. doi:10.1046/j.0022-3042.2002.00760.x
S. Karmakar, N. L. Banik, S. J. Patel and S. K. Ray, “Garlic Compounds Induced Calpain and Intrinsic Caspase Cascade for Apoptosis in Human Malignant Neuroblastoma SH-SY5Y Cells,” Apoptosis, Vol. 12, No. 4, 2007, pp. 671-684. doi:10.1007/s10495-006-0024-x
S. Batra, C. P. Reynolds and B. J. Maurer, “Fenretinide Cytotoxicity for Ewing’s Sarcoma and Primitive Neuroectodermal Tumor Cell Lines Is Decreased by Hypoxia and Synergistically Enhanced by Ceramide Modulators,” Cancer Research, Vol. 64, No. 15, 2004, pp. 5415-5424. doi:10.1158/0008-5472.CAN-04-0377
Y. Li, S. Upadhyay, M. Bhuiyan and F. H. Sarkar, “Induction of Apoptosis in Breast Cancer Cells MDA-MB- 231 by Genistein,” Oncogene, Vol. 18, No. 20, 1999, pp. 3166-3172. doi:10.1038/sj.onc.1202650
S. A. Alhasan, J. F. Ensley and F. H. Sarkar, “Genistein Induced Molecular Changes in a Squamous Cell Carcinoma of the Head and Neck Cell Line,” International Journal of Oncology, Vol. 16, No. 2, 2000, pp. 333-338.
C. Y. Wang, M. W. Mayo, R. G. Korneluk, D. V. Goeddel and A. S. Baldwin Jr., “NF-κB Antiapoptosis: Induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to Suppress Caspase-8 Activation,” Science, Vol. 281, No. 5383, 1999, pp. 1680-1683. doi:10.1126/science.281.5383.1680
E. C. La Casse, S. Baird, R. G. Korneluk and A. E. MacKenzie, “The Inhibitors of Apoptosis (IAPs) and Their Emerging Role in Cancer,” Oncogene, Vol. 17, No. 25, 1998, pp. 3247-3259. doi:10.1038/sj.onc.1202569
A. Büki, D. O. Okonkwo, K. K. Wang and J. T. Povlishock, “Cytochrome c Release and Caspase Activation in Traumatic Axonal Injury,” The Journal of Neuroscience, Vol. 20, No. 8, 2000, pp. 2825-2834.
F. Altznauer, S. Conus, A. Cavalli, G. Folkers and H. U. Simon, “Calpain-1 Regulates Bax and Subsequent Smac- dependent Caspase-3 Activation in Neutrophil Apoptosis,” The Journal of Biological Chemistry, Vol. 279, No. 7, 2004, pp. 5947-5957. doi:10.1074/jbc.M308576200
R. Nath, K. J. Raser, D. Stafford, I. Hajimohammadreza, A. Posner, H. Allen, R. V. Talanian, P. Yuen, R. B. Gilbertsen and K. K. Wang, “Non-Erythroid α-Spectrin Breakdown by Calpain and Interleukin 1β-Converting- -Enzyme-Like Protease(s) in Apoptotic Cells: Contributory Roles of Both Protease Families in Neuronal Apoptosis,” Biochemical Journal, Vol. 319, No. 3, 1996, pp. 683-690.
K. K. Wang, R. Posmantur, R. Nath, K. McGinnis, M. Whitton, R. V. Talanian, S. B. Glantz and J. S. Morrow, “Simultaneous Degradation of II- and II-Spectrin by Caspase 3 (CPP32) in Apoptotic Cells,” The Journal of Biological Chemistry, Vol. 273, No. 35, 1998, pp. 22490- 22497. doi:10.1074/jbc.273.35.22490
S. P. Cregan, V. L. Dawson and R. S. Slack, “Role of AIF in Caspase-Dependent and Caspase-Independent Cell Death,” Oncogene, Vol. 23, No. 16, 2004, pp. 2785-2796. doi:10.1038/sj.onc.1207517
G. Cao, J. Xing, X. Xiao, A. K. Liou, Y. Gao, X. M. Yin, R. S. Clark, S. H. Graham and J. Chen, “Critical Role of Calpain I in Mitochondrial Release of Apoptosis-Inducing Factor in Ischemic Neuronal Injury,” The Journal of Neuroscience, Vol. 27, No. 35, 2007, pp. 9278-9293. doi:10.1523/JNEUROSCI.2826-07.2007
H. Kadara, L. Lacroix, D. Lotan and R. Lotan, “Induction of Endoplasmic Reticulum Stress by the Pro-Apoptotic Retinoid N-(4-Hydroxyphenyl) Retinamide via a Reactive Oxygen Species-Dependent Mechanism in Human Head and Neck Cancer Cells,” Cancer Biology & Therapy, Vol. 6, No. 5, 2007, pp. 705-711. doi:10.4161/cbt.6.5.3963
R. Cuperus, R. Leen, G. A. Tytgat, H. N. Caron and A. B. van Kuilenburg, “Fenretinide Induces Mitochondrial ROS and Inhibits the Mitochondrial Respiratory Chain in Neuroblastoma,” Cellular and Molecular Life Sciences, Vol. 67, No. 5, 2010, pp. 807-816. doi:10.1007/s00018-009-0212-2
J. L. Armstrong, R. Flockhart, G. J. Veal, P. E. Lovat and C. P. Redfern, “Regulation of Endoplasmic Reticulum Stress-Induced Cell Death by ATF4 in Neuroectodermal Tumor Cells,” The Journal of Biological Chemistry, Vol. 285, No. 9, 2010, pp. 6091-6100. doi:10.1074/jbc.M109.014092
S. J. Park, M. J. Kim, Y. K. Kim, S. M. Kim, J. Y. Park and H. Myoung, “Combined Cetuximab and Genistein Treatment Shows Additive Anti-Cancer Effect on Oral Squamous Cell Carcinoma,” Cancer Letters, Vol. 292, No. 1, 2010, pp. 54-63. doi:10.1016/j.canlet.2009.11.004
G. Pagnan, D. Di Paolo, R, Carosio, F. Pastorino, D. Marimpietri, C. Brignole, A. Pezzolo, M. Loi, L. J. Galietta, F. Piccardi, M. Cilli, B. Nico, D. Ribatti, V. Pistoia and M. Ponzoni, “The Combined Therapeutic Effects of Bortezomib and Fenretinide on Neuroblastoma Cells Involve Endoplasmic Reticulum Stress Response,” Clinical Cancer Research, Vol. 15, No. 4, 2009, pp. 1199-209. doi:10.1158/1078-0432.CCR-08-2477
S. Muruganandan and A. E. Cribb, “Calpain-Induced Endoplasmic Reticulum Stress and Cell Death Following Cytotoxic Damage to Renal Cells,” Toxicological Sciences, Vol. 94, No. 1, 2006, pp. 118-128. doi:10.1093/toxsci/kfl084
R. V. Rao, S. Castro-Obregon, H. Frankowski, M. Schuler, V. Stoka, G. del Rio, D. E. Bredesen and H. M. Ellerby, “Coupling Endoplasmic Reticulum Stress to the Cell Death Program. An Apaf-1-Independent Intrinsic Pathway,” Journal of Biological Chemistry, Vol. 277, No. 24, 2002, pp. 21836-21842. doi:10.1074/jbc.M202726200
M. Tiwari, A. Kumar, R. A. Sinha, A. Shrivastava, A. K. Balapure, R. Sharma, V. K. Bajpai, K. Mitra, S. Babu and M. M. Godbole, “Mechanism of 4-HPR-Induced Apoptosis in Glioma Cells: Evidences Suggesting Role of Mitochondrial-Mediated Pathway and Endoplasmic Reticulum Stress,” Carcinogenesis, Vol. 27, No. 10, 2006, pp. 2047-2058. doi:10.1093/carcin/bgl051