Synergistic efficacy of sorafenib and genistein in growth inhibition by down regulating angiogenic and survival factors and increasing apoptosis through upregulation of p53 and p21 in malignant neuroblastoma cells having N-Myc amplification or non-amplification.
Neuroblastoma is an extracranial, solid, and heterogeneous malignancy in children. The conventional therapeutic modalities are mostly ineffective and thus new therapeutic strategies for malignant neuroblastoma are urgently warranted. We examined the synergistic efficacy of combination of sorafenib (SF) and genistein (GST) in human malignant neuroblastoma SK-N-DZ (N-Myc amplified) and SH-SY5Y (N-Myc non-amplified) cell lines. MTT assay showed dose-dependent decrease in cell viability and the combination therapy more prominently inhibited the cell proliferation in both cell lines than either treatment alone. Apoptosis was confirmed morphologically by Wright staining. Flow cytometric analysis of cell cycle phase distribution and Annexin V-FITC/PI staining showed increase in subG1 DNA content and early apoptosis, respectively, after treatment with the combination of drugs. Apoptosis was further confirmed by scanning electron microscopy. Combination therapy showed activation of caspase-8, cleavage of Bid to tBid, increase in p53 and p21 expression, down regulation of anti-apoptotic Mcl-1, and increase in Bax:Bcl-2 ratio to trigger apoptosis. Down regulation of MDR, hTERT, N-Myc, VEGF, FGF-2, NF-κB, p-Akt, and c-IAP2 indicated suppression of angiogenic and survival pathways. Mitochondrial release of cytochrome c and Smac into cytosol indicated involvement of mitochondia in apoptosis. Increases in proteolytic activities of calpain and caspase-3 were also confirmed. Our results suggested that combination of SF and GST inhibited angiogenic and survival factors and increased apoptosis via receptor and mitochondria mediated pathways in both neuroblastoma SK-N-DZ and SH-SY5Y cell lines. Thus, this combination of drugs could be a potential therapeutic strategy against human malignant neuroblastoma cells having N-Myc amplification or non-amplification.
Roy Choudhury
Subhasree
S
Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Building 2, Room C11, 6439 Garners Ferry Road, Columbia, SC 29209, USA.
Karmakar
Surajit
S
Banik
Naren L
NL
Ray
Swapan K
SK
D043925:Angiogenesis Inducing Agents / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects*; D001557:Benzenesulfonates / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects; D004357:Drug Synergism; D005434:Flow Cytometry; D005784:Gene Amplification / Q000187:drug effects; D019833:Genistein / Q000494:pharmacology*; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009447:Neuroblastoma / Q000188:drug therapy / Q000235:genetics / Q000473:pathology* / Q000648:ultrastructure; D009536:Niacinamide / Q000031:analogs & derivatives; D010671:Phenylurea Compounds; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D011725:Pyridines / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D000077157:Sorafenib; D019098:Telomerase / Q000378:metabolism; D016896:Treatment Outcome; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D043925:Angiogenesis Inducing Agents; D001557:Benzenesulfonates; D050759:Cyclin-Dependent Kinase Inhibitor p21; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D010671:Phenylurea Compounds; D019253:Proto-Oncogene Proteins c-bcl-2; D016271:Proto-Oncogene Proteins c-myc; D011725:Pyridines; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D009536:Niacinamide; D000077157:Sorafenib; D019833:Genistein; C509186:TERT protein, human; D019098:Telomerase; D053148:Caspase 3