Rottlerin induces autophagy and apoptotic cell death through a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells: the protective role of autophagy in apoptosis.
Rottlerin is widely used as a protein kinase C-delta inhibitor. Recently, several reports have shown the possible apoptosis-inducing effect of rottlerin in some cancer cell lines. Here we report that rottlerin induces not only apoptosis but also autophagy via a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells. Rottlerin treatment induced a dose- and time-dependent inhibition of cell growth, and cytoplasmic vacuolations were markedly shown. These vacuoles were identified as acidic autolysosomes by electron microscopy, acidic vesicular organelle (AVO) staining and transfection of green fluorescent protein-LC3. The LC3-II protein level also increased after treatment with rottlerin. Prolonged exposure to rottlerin eventually caused apoptosis via loss of mitochondrial membrane potential and translocation of AIF from mitochondria to the nucleus. However, the activities of caspase-3, -8 and -9 were not changed, and PARP did not show signs of cleavage. Interestingly, the pretreatment of cells with a specific inhibitor of autophagy (3-methyladenine) accelerated rottlerin-induced apoptosis as revealed by an analysis of the subdiploid fraction and TUNEL assay. Nevertheless, the knockdown of PKC-delta by RNA interference neither affected cell growth nor acidic vacuole formation. Similarly, rottlerin-induced cell death was not prevented by PKC-delta overexpression. Taken together, these findings suggest that rottlerin induces early autophagy and late apoptosis in a PKC-delta-independent manner, and the rottlerin-induced early autophagy may act as a survival mechanism against late apoptosis in HT1080 human fibrosarcoma cells.
Song
Kyoung-Sub
KS
Department of Biochemistry, Chungnam National University, Munhwa-dong, Joong-gu, Daejeon, Korea.
Kim
Jong-Seok
JS
Yun
Eun-Jin
EJ
Kim
Young-Rae
YR
Seo
Kang-Sik
KS
Park
Ji-Hoon
JH
Jung
Yeon-Joo
YJ
Park
Jong-Il
JI
Kweon
Gi-Ryang
GR
Yoon
Wan-Hee
WH
Lim
Kyu
K
Hwang
Byung-Doo
BD
D000098:Acetophenones / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001343:Autophagy / Q000187:drug effects* / Q000502:physiology; D001578:Benzopyrans / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D005354:Fibrosarcoma / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology*; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051745:Protein Kinase C-delta / Q000037:antagonists & inhibitors* / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology
D000098:Acetophenones; D001578:Benzopyrans; C085746:rottlerin; D051745:Protein Kinase C-delta