title: In vitro selected RNA aptamer recognizing glutathione induces ROS mediated apoptosis in the human breast cancer cell line MCF 7.
abstract: Glutathione (GSH) is an abundant natural tripeptide with antioxidant properties. Under different conditions, it can play protective as well as pathogenic roles. The redox state of the cell has an important role in the induction of apoptosis. Elevated level of glutathione in cancer cells provides resistance to a number of chemotherapeutic drugs. Inhibition of glutathione synthesis sensitizes the cells for apoptosis and enhances the activity of chemotherapeutic drugs. We have selected GSH-binding RNA aptamers by employing in vitro selection protocol SELEX. The Kd value of these aptamers with respect to GSH were determined by surface plasmon resonance (SPR) analysis and isocratic affinity chromatography. Two aptamers GSHapt 8.17 (class-III) and GSHapt 5.39 (class-IV) had Kd values of 4.18 and 4.89 x 10(-8) M, respectively and GSHapt class-I had a Kd value of 1.2 x 10(-6) M. CD spectra suggested conformational change in aptamers upon GSH binding. Cultured breast cancer cells (MCF7) responded to expression of GSH aptamers by accumulating ROS and undergoing morphological transition, nuclear condensation, and DNA fragmentation, with concurrent depletion of cellular GSH and activation of caspase 3 eventually leading to apoptosis. DTT and caspase-3 inhibitor partially rescued aptamer induced apoptosis. These aptamers exhibit high specificity to GSH over non specific competitor. The same aptamers did not induce apoptosis in 293T cells. The kinetic properties and pro-apoptotic effects suggest that glutathione-binding RNA aptamer could be developed into an effective anti-cancer chemotherapeutic agent.
authors.affiliation: Applied Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
authors.forename: Jyoti
authors.initials: J
authors.lastname: Bala
authors.forename: Ashima
authors.initials: A
authors.lastname: Bhaskar
authors.forename: Akhil
authors.initials: A
authors.lastname: Varshney
authors.forename: Abhay Kumar
authors.initials: AK
authors.lastname: Singh
authors.forename: Sharmistha
authors.initials: S
authors.lastname: Dey
authors.forename: Pramod
authors.initials: P
authors.lastname: Yadava
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000494:pharmacology*; D001483:Base Sequence; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D053938:DNA Fragmentation; D005260:Female; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000032:analysis / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D014162:Transfection
chemical_list: D000964:Antimetabolites, Antineoplastic; D052157:Aptamers, Nucleotide; D017382:Reactive Oxygen Species; C505407:CASP3 protein, human; D053148:Caspase 3; D005978:Glutathione