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.