Prediction and validation of apoptosis through cytochrome P450 activation by benzo[a]pyrene.
Polycyclic aromatic hydrocarbons (PAHs) processed by cytochrome P450 (CYP450) during metabolism is well reported to induce carcinogenesis. The present study has developed a new approach to examine apoptotic activity of a known PAH called benzo[a]pyrene (B[a]P), using protein-ligand and protein-protein interaction through in silico approach, followed by in vitro validation. In silico study showed that the conformational changes and energies involved in the binding of B[a]P to CYP1B1 was crucial with its target proteins. The data showed that activated B[a]P had high affinity to bind with aryl hydrocarbon receptor (AhR) with binding energy of -601.97kcal/mol. Interestingly, B[a]P-CYP1B1 complex showed strong binding affinity for caspase-8, -9, -3 with binding energy of -625.5, -479.3 and -514.2kcal/mol respectively. Moreover, the docking of specific caspase inhibitors in the complex showed weak interaction with low binding energy value as compared to B[a]P-CYP1B1 caspase complexes. To validate our in silico work, we showed B[a]P treated HaCaT cells triggered apoptosis with increase in caspase 8, caspase 9 and caspase 3/7 level. Further, in vitro work confirmed that B[a]P induced apoptosis was significantly suppressed in Ac-DEVD-CMK pre-treated cells. In addition, knockdown of CYP1B1 suppressed B[a]P induced apoptosis in HaCaT cells confirming a pivotal role of CYP1B1 in B[a]P induced apoptosis. Interestingly, through in silico modeling, we screened clotrimazole as a potent CYP1B1 inhibitor which completely inhibited B[a]P mediated activation. This hypothesis was validated by MTT assay, caspase activation measurement and showed remarkable inhibition of B[a]P induced cell death; thereby, highlighting a potent therapeutic role for industrial pollution associated diseases.
Department of Life Science, National Institute of Technology, Rourkela, Odisha, India.
Durgesh Nandini
DN
Das
Department of Life Science, National Institute of Technology, Rourkela, Odisha, India.
Prashanta Kumar
PK
Panda
Department of Life Science, National Institute of Technology, Rourkela, Odisha, India.
Subhadip
S
Mukhopadhyay
Department of Life Science, National Institute of Technology, Rourkela, Odisha, India.
Niharika
N
Sinha
Department of Life Science, National Institute of Technology, Rourkela, Odisha, India.
Bibekanand
B
Mallick
Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal, India.
Birendra
B
Behera
Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal, India.
Tapas K
TK
Maiti
Department of Life Science, National Institute of Technology, Rourkela, Odisha, India. Electronic address: sujitb@nitrkl.ac.in.
Sujit K
SK
Bhutia
D017209:Apoptosis / Q000187:drug effects*; D001189:Aryl Hydrocarbon Hydroxylases / Q000172:deficiency / Q000378:metabolism; D001564:Benzo(a)pyrene / Q000494:pharmacology*; D061945:Caspase Inhibitors / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D003022:Clotrimazole / Q000494:pharmacology; D065633:Cytochrome P-450 CYP1B1; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D054730:Protein Interaction Domains and Motifs / Q000187:drug effects; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism
D061945:Caspase Inhibitors; D018336:Receptors, Aryl Hydrocarbon; D001564:Benzo(a)pyrene; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D065633:Cytochrome P-450 CYP1B1; D020169:Caspases; D003022:Clotrimazole
Apoptosis; Aryl hydrocarbon receptor; Benzo[a]pyrene; Cytochrome P450; Ligand–protein; Protein–protein