title: Investigation of the mechanism and apoptotic pathway induced by 4β cinnamido linked podophyllotoxins against human lung cancer cells A549.
abstract: Apoptosis is essential for normal development and the maintenance of homeostasis. It plays a necessary role to protect against carcinogenesis by eliminating damaged cells. Many studies have demonstrated that the dysregulation of apoptosis results in cancer and this provides an approach to develop therapeutic agents via inducing apoptosis. In our previous studies 4β-cinnamido linked podophyllotoxin conjugates were synthesized and evaluated for their cytotoxic activity in a panel of five human cancer cell lines and the new molecules like 17a and 17f were considered as potential leads. The cytotoxic activity was comparable to etoposide. These observations prompted us to investigate the mechanism underplaying the cytotoxic activity and apoptotic pathway induced by these compounds in human lung cancer cells A459. The results of the present study revealed that these compounds exhibited DNA topoisomerase IIα inhibition and induced mitochondrial mediated apoptosis. It was further confirmed by Mitochondrial membrane potential, Cytochrome c release, cleavage of poly (ADP-ribose) polymerase (PARP), Reactive oxygen species (ROS) generation, regulation of antiapoptotic protein Bcl-2 and pro apoptotic protein Bax studied by Western blot analysis. Annexin V-FITC assay also suggested that these compounds induced cell death by apoptosis. Pretreatment with N-acetyl-L-cysteine (NAC) prevented the generation of ROS. Further, pretreatment with NAC significantly inhibited 17a and 17f induced apoptosis, suggesting that ROS are the key mediators for 17a and 17f induced apoptosis. These data indicate that these compounds might induce apoptosis in A549 cells through a ROS mediated mitochondrial dysfunction pathway. Moreover, these compounds did not significantly inhibit the noncancerous human embryonic kidney cells, HEK-293. Docking studies also elucidate the potential of these molecules to bind to the DNA topoisomerase II. Podophyllotoxin analogs were investigated for their mechanism and apoptotic pathway against lung cancer cell line, A549. These podophyllotoxin analogs inhibited DNA topoisomerase IIα and induced mitochondrial mediated apoptosis in lung cancer cell line, A549. Western blot analysis suggested that these compounds inhibited the DNA topoisomerase IIα. Studies like, Measurement of mitochondrial membrane potential (∆Ψm), Generation of intracellular reactive oxygen species (ROS) and Annexin V-FITC assay suggested that these compounds induced mitochondrial mediated apoptosis. Pretreatment with N-acetyl-L-cysteine (NAC) suggested that ROS plays a role in 17a and 17f induced apoptosis. Further the apoptotic effect of these compounds was confirmed by western blot analysis of pro apoptotic protein Bax and antiapoptotic protein Bcl-2, Cytochrome c release and cleavage of poly (ADP-ribose) polymerase (PARP). Moreover, these compounds did not significantly inhibit the noncancerous human embryonic kidney cells, HEK-293.
authors.affiliation: Medicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500 007, India. ahmedkamal@iict.res.in.|Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Hyderabad, 500 037, India. ahmedkamal@iict.res.in.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Kamal
authors.affiliation: Medicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500 007, India.
authors.forename: V Lakshma
authors.initials: VL
authors.lastname: Nayak
authors.affiliation: Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Hyderabad, 500 037, India.
authors.forename: Chandrakant
authors.initials: C
authors.lastname: Bagul
authors.affiliation: Medicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500 007, India.
authors.forename: M V P S
authors.initials: MV
authors.lastname: Vishnuvardhan
authors.affiliation: Medicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500 007, India.
authors.forename: Adla
authors.initials: A
authors.lastname: Mallareddy
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D015394:Molecular Structure; D011034:Podophyllotoxin / Q000737:chemistry / Q000494:pharmacology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D017382:Reactive Oxygen Species; D045304:Cytochromes c; D011065:Poly(ADP-ribose) Polymerases; D011034:Podophyllotoxin
keywords: Annexin V-FITC; Anticancer activity; Apoptosis; Topoisomerase IIα and IIβ; Western blot analysis