Gemcitabine Induces Poly (ADP-Ribose) Polymerase-1 (PARP-1) Degradation through Autophagy in Pancreatic Cancer

<div><p>Poly (ADP-ribose) polymerase-1 (PARP-1) and autophagy play increasingly important roles in DNA damage repair and cell death. Gemcitabine (GEM) remains the first-line chemotherapeutic drug for pancreatic cancer (PC). However, little is known about the relationship between PARP-1 expression and autophagy in response to GEM. Here we demonstrate that GEM induces DNA-damage response and degradation of mono-ADP ribosylated PARP-1 through the autophagy pathway in PC cells, which is rescued by inhibiting autophagy. Hypoxia and serum starvation inhibit autophagic activity due to abrogated GEM-induced mono-ADP-ribosylated PARP-1 degradation. Activation of extracellular regulated protein kinases (ERK) induced by serum starvation shows differences in intracellular localization as well as modulation of autophagy and PARP-1 degradation in GEM-sensitive KLM1 and -resistant KLM1-R cells. Our study has revealed a novel role of autophagy in PARP-1 degradation in response to GEM, and the different impacts of MEK/ERK signaling pathway on autophagy between GEM-sensitive and -resistant PC cells.</p></div

Serum starvation induces activation and different localization of extracellular ERK between KLM1 and KLM1-R cells.

<p>(A) KLM1 and KLM1-R cells were cultured in medium with or without FBS or exposed to 10 μg/mL of GEM for 24 h. Cell lysates were resolved by SDS-PAGE and probed with specific antibodies against p-ERK and ERK. (B) and (C) The indicated cells were stained with specific antibodies against p-ERK, Hsp27 and LC3A/B after cells were cultured in medium with or without FBS for 24 h. DAPI: blue and p-ERK: red in (B) and LC3A/B: green and Hsp27: red in (C). Scale bar, 20 μm.</p

Hypoxia suppresses autophagy and GEM-induced PARP-1 degradation.

<p>(A) KLM1 and KLM1-R cells were cultured in normal conditions or 1% O₂ hypoxia for 24 hours, and then cell lysates were resolved by SDS-PAGE and probed with specific antibodies. (B) The indicated cells were cultured in normal conditions or 1% O₂ hypoxia together with 10 μg/mL of GEM for 24 hours. Cell lysates were resolved by SDS-PAGE and probed with specific antibodies. An arrow head indicates the mono-ADP ribosylated form of PARP-1. Arrows indicate the position area of cleaved caspase-3. The expression of PARP-1 was confirmed repeatedly by a distinct PARP-1 antibody described in Materials.</p

Serum starvation suppresses GEM-induced PARP-1 degradation through inhibition of autophagy via the ERK signaling pathway.

<p>(A) KLM1 and KLM1-R cells were exposed to 10 μg/mL of GEM in the presence or absence of 20 μM of U0126 for the indicated time courses. Cell lysates were resolved by SDS-PAGE and probed with specific antibodies against p-ERK and LC3A/B. (B) and (C) KLM1 and KLM1-R cells were cultured in the medium with or without FBS and meanwhile exposed to either or both GEM and U0126 at the indicated concentration. Cell lysates were resolved by SDS-PAGE and probed with specific antibodies. The arrow head indicates the mono-ADP ribosylated form of PARP-1. Arrows indicate the position area of cleaved caspase-3. The expression of PARP-1 was confirmed repeatedly by a distinct PARP-1 antibody described in Materials.</p

GEM induces autophagy in PC cells.

<p>(A) The expression of Hsp27 was tested by western blot and the relative intensity was measured by student-<i>t</i> test (n = 3) (B) KLM1 and KLM1-R cells were lysed and resolved by SDS-PAGE and probed with specific antibodies. Actin was used to normalize the loading levels of protein. (C) The indicated cells were treated with 100 μg/mL of GEM for 5 h and the expression of LC3A/B and formation of LC3-positive autophagosomes were examined by confocal microscopy. LC3A/B: green and DAPI: blue. Arrows indicate the autophagosome. Scale bar, 20 μm.</p

GEM down-regulates mono-ADP ribosylated PARP-1 in a caspase-independent manner.

<p>(A) KLM1 and KLM1-R cells were treated by GEM with the indicated concentrations for 24 h. Cell lysates were resolved in SDS-PAGE and probed with specific antibodies. The expression of PARP-1 was confirmed repeatedly by a distinct PARP-1 antibody described in Materials. An arrow head indicated the mono-ADP ribosylated form of PARP-1. Arrows indicated the position area of cleaved caspase-3. (B) The indicated cells were treated as in (A) and then stained using a caspases 3/7 assay kit (A). Caspase 3/7 activity was tested and measured by confocal microscopy and Image J. N.S., non significant. Error bars, SD.</p

GEM suppresses mono-ADP ribosylated PARP-1 expression via autophagy.

<p>(A) KLM1 and KLM1-R cells were treated with 100 μg/mL of GEM for 5 h. Treated cells were stained with specific antibodies against LC3A/B and PARP-1 and then detected by confocal microscopy. LC3A/B: green, PARP-1: red and DAPI: blue. Scale bar, 20 μm. Arrows indicate the yellow staining of co-localizations between autophagosome and PARP-1. (B) KLM1 cells were exposed to GEM for 24 h after LC3B knockdown. (C) KLM1 and KLM1-R cells were exposed to GEM for 24 h in the present or absent of either PMSF or wortmannin at the indicated concentrations. Cell lysates were resolved by SDS-PAGE and probed with specific antibodies against to PARP-1. The arrow head indicates the mono-ADP ribosylated form of PARP-1. The expression of PARP-1 was confirmed repeatedly by a distinct PARP-1 antibody described in Materials.</p