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Neuregulin promotes incomplete autophagy of prostate cancer cells that is independent of mTOR pathway inhibition.

Schmukler E, Shai B, Ehrlich M, Pinkas-Kramarski R - PLoS ONE (2012)

Bottom Line: It was also demonstrated that PI3K inhibitors repressed this cell death suggesting that in androgen deprived LNCaP cells, NRG activates a PI3K-dependent pathway associated with cell death.Also, in cells overexpressing Bcl-2 or cells expressing sh-RNA against Beclin 1, the effects of NRG, namely induction of autophagy and cell death, were inhibited.These effects of NRG are mediated by signaling pathway that activates JNK and Beclin 1, but is independent of mTOR inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Tel-Aviv University, Ramat-Aviv, Israel.

ABSTRACT

Background: Growth factors activating the ErbB receptors have been described in prostate tumors. The androgen dependent prostate cancer cell line, LNCaP, expresses the ErbB-1, ErbB-2 and ErbB-3 receptor tyrosine kinases. Previously, it was demonstrated that NRG activates ErbB-2/ErbB-3 heterodimers to induce LNCaP cell death, whereas, EGF activates ErbB-1/ErbB-1 or ErbB-1/ErbB-2 dimers to induce cell growth and survival. It was also demonstrated that PI3K inhibitors repressed this cell death suggesting that in androgen deprived LNCaP cells, NRG activates a PI3K-dependent pathway associated with cell death.

Methodology/principal findings: In the present study we demonstrate that NRG induces autophagy in LNCaP cells, using LC3 as a marker. However, the autophagy induced by NRG may be incomplete since p62 levels elevate. We also demonstrated that NRG- induced autophagy is independent of mammalian target of rapamycin (mTOR) inhibition since NRG induces Akt and S6K activation. Interestingly, inhibition of reactive oxygen species (ROS) by N-acetylcysteine (NAC), inhibited NRG-induced autophagy and cell death. Our study also identified JNK and Beclin 1 as important components in NRG-induced autophagy and cell death. NRG induced elevation in JNK phosphorylation that was inhibited by NAC. Moreover, inhibitor of JNK inhibited NRG-induced autophagy and cell death. Also, in cells overexpressing Bcl-2 or cells expressing sh-RNA against Beclin 1, the effects of NRG, namely induction of autophagy and cell death, were inhibited.

Conclusions/significance: Thus, in LNCaP cells, NRG-induces incomplete autophagy and cell death that depend on ROS levels. These effects of NRG are mediated by signaling pathway that activates JNK and Beclin 1, but is independent of mTOR inhibition.

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Related in: MedlinePlus

NRG-induced autophagy is incomplete compared to starvation-induced autophagy.(A) LNCaP cells stably expressing LC3-GFP were treated with 100 ng/ml NRG for 24 h or incubated with EBSS medium for the indicated time periods. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-GFP antibody. (B) LNCaP cells stably expressing LC3-GFP were treated with 100 ng/ml NRG for 24 h or incubated with EBSS medium for 2 and 4 h. Treatments were performed in the presence or absence of 10 nM bafilomycin-A1 (Bafilo-A1). Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-GFP antibody. Upper panel, representative blot. Lower panel, densitometric analysis is presented as fold induction over the control untreated cells (left graph; *, p<0.05 and **, p<0.02 compared with the control) or as difference between the measured values with or without 10 nM Bafilo-A1 in each group (right graph; *, p<0.05 and **, p<0.02 compared with NRG tretment) (n = 3; means ± S.D).
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pone-0036828-g002: NRG-induced autophagy is incomplete compared to starvation-induced autophagy.(A) LNCaP cells stably expressing LC3-GFP were treated with 100 ng/ml NRG for 24 h or incubated with EBSS medium for the indicated time periods. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-GFP antibody. (B) LNCaP cells stably expressing LC3-GFP were treated with 100 ng/ml NRG for 24 h or incubated with EBSS medium for 2 and 4 h. Treatments were performed in the presence or absence of 10 nM bafilomycin-A1 (Bafilo-A1). Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-GFP antibody. Upper panel, representative blot. Lower panel, densitometric analysis is presented as fold induction over the control untreated cells (left graph; *, p<0.05 and **, p<0.02 compared with the control) or as difference between the measured values with or without 10 nM Bafilo-A1 in each group (right graph; *, p<0.05 and **, p<0.02 compared with NRG tretment) (n = 3; means ± S.D).

Mentions: NRG treatment induces autophagy but also leads to an increase in p62 levels, indicating that the autophagy induced by NRG is incomplete (Figure 1). To further confirm these results, LNCaP cells stably expressing LC3-GFP fusion protein were either treated with NRG for 24 h or incubated with EBSS medium for several hours. Cell lysates were immunoblotted with anti-GFP antibody to detect the levels of GFP-tagged LC3-I and LC3-II. As shown in figure 2A, both EBSS and NRG induce autophagy, as judged by the increase of LC3-II-GFP levels compared to the untreated cells. However, in cells incubated with EBSS, LC3-II-GFP levels decreases over time, while in cells treated with NRG the levels of LC3-II-GFP are relatively high even after 24 h incubation. Furthermore, in the presence of bafilomycin-A1 (inhibitor of autophagosome-lysosome fusion) the accumulation of LC3-II-GFP is significantly higher in the EBSS treated cells compared to NRG treated cells (Figure 2B). Taken together, these finding indicate that NRG-induced autophagy is incomplete.


Neuregulin promotes incomplete autophagy of prostate cancer cells that is independent of mTOR pathway inhibition.

Schmukler E, Shai B, Ehrlich M, Pinkas-Kramarski R - PLoS ONE (2012)

NRG-induced autophagy is incomplete compared to starvation-induced autophagy.(A) LNCaP cells stably expressing LC3-GFP were treated with 100 ng/ml NRG for 24 h or incubated with EBSS medium for the indicated time periods. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-GFP antibody. (B) LNCaP cells stably expressing LC3-GFP were treated with 100 ng/ml NRG for 24 h or incubated with EBSS medium for 2 and 4 h. Treatments were performed in the presence or absence of 10 nM bafilomycin-A1 (Bafilo-A1). Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-GFP antibody. Upper panel, representative blot. Lower panel, densitometric analysis is presented as fold induction over the control untreated cells (left graph; *, p<0.05 and **, p<0.02 compared with the control) or as difference between the measured values with or without 10 nM Bafilo-A1 in each group (right graph; *, p<0.05 and **, p<0.02 compared with NRG tretment) (n = 3; means ± S.D).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351469&req=5

pone-0036828-g002: NRG-induced autophagy is incomplete compared to starvation-induced autophagy.(A) LNCaP cells stably expressing LC3-GFP were treated with 100 ng/ml NRG for 24 h or incubated with EBSS medium for the indicated time periods. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-GFP antibody. (B) LNCaP cells stably expressing LC3-GFP were treated with 100 ng/ml NRG for 24 h or incubated with EBSS medium for 2 and 4 h. Treatments were performed in the presence or absence of 10 nM bafilomycin-A1 (Bafilo-A1). Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-GFP antibody. Upper panel, representative blot. Lower panel, densitometric analysis is presented as fold induction over the control untreated cells (left graph; *, p<0.05 and **, p<0.02 compared with the control) or as difference between the measured values with or without 10 nM Bafilo-A1 in each group (right graph; *, p<0.05 and **, p<0.02 compared with NRG tretment) (n = 3; means ± S.D).
Mentions: NRG treatment induces autophagy but also leads to an increase in p62 levels, indicating that the autophagy induced by NRG is incomplete (Figure 1). To further confirm these results, LNCaP cells stably expressing LC3-GFP fusion protein were either treated with NRG for 24 h or incubated with EBSS medium for several hours. Cell lysates were immunoblotted with anti-GFP antibody to detect the levels of GFP-tagged LC3-I and LC3-II. As shown in figure 2A, both EBSS and NRG induce autophagy, as judged by the increase of LC3-II-GFP levels compared to the untreated cells. However, in cells incubated with EBSS, LC3-II-GFP levels decreases over time, while in cells treated with NRG the levels of LC3-II-GFP are relatively high even after 24 h incubation. Furthermore, in the presence of bafilomycin-A1 (inhibitor of autophagosome-lysosome fusion) the accumulation of LC3-II-GFP is significantly higher in the EBSS treated cells compared to NRG treated cells (Figure 2B). Taken together, these finding indicate that NRG-induced autophagy is incomplete.

Bottom Line: It was also demonstrated that PI3K inhibitors repressed this cell death suggesting that in androgen deprived LNCaP cells, NRG activates a PI3K-dependent pathway associated with cell death.Also, in cells overexpressing Bcl-2 or cells expressing sh-RNA against Beclin 1, the effects of NRG, namely induction of autophagy and cell death, were inhibited.These effects of NRG are mediated by signaling pathway that activates JNK and Beclin 1, but is independent of mTOR inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Tel-Aviv University, Ramat-Aviv, Israel.

ABSTRACT

Background: Growth factors activating the ErbB receptors have been described in prostate tumors. The androgen dependent prostate cancer cell line, LNCaP, expresses the ErbB-1, ErbB-2 and ErbB-3 receptor tyrosine kinases. Previously, it was demonstrated that NRG activates ErbB-2/ErbB-3 heterodimers to induce LNCaP cell death, whereas, EGF activates ErbB-1/ErbB-1 or ErbB-1/ErbB-2 dimers to induce cell growth and survival. It was also demonstrated that PI3K inhibitors repressed this cell death suggesting that in androgen deprived LNCaP cells, NRG activates a PI3K-dependent pathway associated with cell death.

Methodology/principal findings: In the present study we demonstrate that NRG induces autophagy in LNCaP cells, using LC3 as a marker. However, the autophagy induced by NRG may be incomplete since p62 levels elevate. We also demonstrated that NRG- induced autophagy is independent of mammalian target of rapamycin (mTOR) inhibition since NRG induces Akt and S6K activation. Interestingly, inhibition of reactive oxygen species (ROS) by N-acetylcysteine (NAC), inhibited NRG-induced autophagy and cell death. Our study also identified JNK and Beclin 1 as important components in NRG-induced autophagy and cell death. NRG induced elevation in JNK phosphorylation that was inhibited by NAC. Moreover, inhibitor of JNK inhibited NRG-induced autophagy and cell death. Also, in cells overexpressing Bcl-2 or cells expressing sh-RNA against Beclin 1, the effects of NRG, namely induction of autophagy and cell death, were inhibited.

Conclusions/significance: Thus, in LNCaP cells, NRG-induces incomplete autophagy and cell death that depend on ROS levels. These effects of NRG are mediated by signaling pathway that activates JNK and Beclin 1, but is independent of mTOR inhibition.

Show MeSH
Related in: MedlinePlus