A Novel Anticancer Agent, 8-Methoxypyrimido[4',5':4,5]thieno(2,3-b) Quinoline-4(3H)-One Induces Neuro 2a Neuroblastoma Cell Death through p53-Dependent, Caspase-Dependent and -Independent Apoptotic Pathways.

Upasana Sahu,Gopal M Advirao,Sathees C Raghavan,Ranjit K Giri,Pankaj S Ghate,Himakshi Sidhar,Hari Koul

doi:10.1371/journal.pone.0066430

Upasana Sahu, Gopal M Advirao + Show 5 more

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https://doi.org/10.1371/journal.pone.0066430

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Abstract

Neuroblastoma is the most common cancer in infants and fourth most common cancer in children. Despite recent advances in cancer treatments, the prognosis of stage-IV neuroblastoma patients continues to be dismal which warrant new pharmacotherapy. A novel tetracyclic condensed quinoline compound, 8-methoxypyrimido [4′,5′:4,5]thieno(2,3-b) quinoline-4(3H)-one (MPTQ) is a structural analogue of an anticancer drug ellipticine and has been reported to posses anticancer property. Study on MPTQ on neuroblastoma cells is very limited and mechanisms related to its cytotoxicity on neuroblastoma cells are completely unknown. Here, we evaluated the anticancer property of MPTQ on mouse neuro 2a and human SH-SY5Y neuroblastoma cells and investigated the mechanisms underlying MPTQ-mediated neuro 2a cell death. MPTQ-mediated neuro 2a and SH-SY5Y cell deaths were found to be dose and time dependent. Moreover, MPTQ induced cell death reached approximately 99.8% and 90% in neuro 2a and SH-SY5Y cells respectively. Nuclear oligonucleosomal DNA fragmentation and Terminal dUTP Nick End Labelling assays indicated MPTQ-mediated neuro 2a cell death involved apoptosis. MPTQ-mediated apoptosis is associated with increased phosphorylation of p53 at Ser15 and Ser20 which correlates with the hyperphosphorylation of Ataxia-Telangiectasia mutated protein (ATM). Immunocytochemical analysis demonstrated the increased level of Bax protein in MPTQ treated neuro 2a cells. MPTQ-mediated apoptosis is also associated with increased activation of caspase-9, -3 and -7 but not caspase-2 and -8. Furthermore, increased level of caspase-3 and cleaved Poly (ADP Ribose) polymerase were observed in the nucleus of MPTQ treated neuro 2a cells, suggesting the involvement of caspase-dependent intrinsic but not extrinsic apoptotic pathway. Increased nuclear translocation of apoptosis inducing factor suggests additional involvement of caspase-independent apoptosis pathway in MPTQ treated neuro 2a cells. Collectively, MPTQ-induced neuro 2a cell death is mediated by ATM and p53 activation, and Bax-mediated activation of caspase-dependent and caspase-independent mitochondrial apoptosis pathways.

Highlights

Neuroblastoma is an aggressive, heterogeneous and most common extra cranial childhood tumour affecting approximately 1 in 7000 children [1,2]
The results from the present study clearly demonstrated that, MPTQ, a quinoline derivative compound, a structural analogue of ellipticine is cytotoxic to mouse neuro 2a neuroblastoma cells and to human SH-SY5Y neuroblastoma cells in a dose and time-dependent manner
Our results for the first time illustrated the mechanisms associated with MPTQ-mediated cytotoxicity in neuroblastoma cells

Summary

Introduction

Neuroblastoma is an aggressive, heterogeneous and most common extra cranial childhood tumour affecting approximately 1 in 7000 children [1,2]. It originates from neuronal precursor cells of sympathetic lineage and gives rise to tumours along sympathetic nervous system [3,4]. 2 out of 101 neuroblastoma patients with stage III or IV remain disease free and in others, disease relapsed soon after completing the therapy

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