Abstract:
Camptothecin (CPT) is known to selectively inhibit the nuclear enzyme DNA
topoisomerase I which catalyzes the relaxation of negatively supercoiled DNA and
thus leads to cell cycle arrest and apoptosis through DNA damages. However,
whether CPT induces endoplasmic reticulum (ER) stress and autophagy has not been
clearly understood. The LNCaP cells were cultured at 37°C in a 5% CO2-humidified
incubator and maintained in RPMI 1640 culture medium containing 10% FBS and
antibiotics mixtures. Total RNA was isolated from LNCaP cells using Easy-Blue
total RNA extraction kit (iNtRON Biotechnology, Sungnam, Republic of Korea.)
according to the manufacturer′s instruction. Total cell extracts were separated on
polyacrylamide gels and standard procedures were used to transfer them to the
nitrocellulose membranes. Cells were seeded on a 24-well plate at a density of
1 × 105 cells/ml and transfected c-Myc-, JNK-, and eIF2α-specific silencing RNA
(siRNA, Santa Cruz Biotechnology) for 24 h. Present study first reported that CPT
enhanced DNA-binding activity of c-Myc in LNCaP cells according to electronic
mobility shift assay and transient knockdown of c-Myc completely abrogated
reactive oxygen species (ROS) generation which was resulting in accumulation of
ER stress-regulating proteins such as PERK, eIF2α, ATF4, and CHOP. These
observations suggests that CPT-induced c-Myc triggered ER stress along with the
PERK-eIF2α-ATF4-CHOP pathway by increasing ROS generation. Moreover, CPT
promoted formation of autophagy accompanied by increasing autophagic proteins
such as beclin-1 and Atg7. Transfection of eIF2α-targeted siRNA attenuated CPTinduced
beclin-1 and Atg7 expression. Treatment with autophagy inhibitors such as
3-methyladenine and bafilomycin A1 downregulated relative cell viability in
response to CPT, which indicate that CPT induces ER stress-mediated cytoprotective
autophagy. Additionally, CPT significantly induced AMPK phosphorylation as a
result of intracellular Ca2+ release. Moreover, CPT phosphorylated JNK and
activated DNA-binding activity of AP-1, and knockdown of JNK abolished the
expression level of beclin-1 and Atg7, implying that the JNK-AP-1 pathway is a
potent mediator on CPT-induced autophagy. Our findings indicate that CPT
promotes ROS-mediated ER stress through the PERK-eIF2α-ATF-4-CHOP
pathway, which enhances cytoprotective autophagy, resulting from the Ca2+-AMPK
pathway and the JNK-AP-1 pathway.
