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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 Beclin-1 dependent.(A) LNCaP cells were transfected with Beclin 1 sh-RNA (3 µg) or control scrambled sh-RNA (3 µg) and incubated in normal medium for 72 h. The cells were then treated with 100 ng/ml NRG for additional 24 h. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3 and anti-Beclin 1 antibodies. Left panel, representative experiment is shown. Right panel, quantification of the results is shown. The results are presented as fold induction compared to the control untreated cells (n = 3; means ± S.D; *p<0.05). (B) Naïve or Bcl-2-GFP stably expressing LNCaP cells were treated with 100 ng/ml NRG for the indicated time periods. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3 and anti-Bcl-2 antibodies. Left panel, representative blot is shown. Right panel, quantification of the results is presented as fold induction compared to the control untreated cells (n = 3; means ± S.D; *p<0.05). (C) Naïve and Bcl-2-GFP stably expressing LNCaP cells were tested for cell viability using the methylene blue staining assay. Cells were treated with 100 ng/ml NRG and the methylene blue assay was performed 60 h later. Results are presented as % of control, and are the mean ± S.D of 4–6 determinations (**p<0.0001). This experiment was repeated three times with similar results.
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pone-0036828-g007: NRG-induced autophagy is Beclin-1 dependent.(A) LNCaP cells were transfected with Beclin 1 sh-RNA (3 µg) or control scrambled sh-RNA (3 µg) and incubated in normal medium for 72 h. The cells were then treated with 100 ng/ml NRG for additional 24 h. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3 and anti-Beclin 1 antibodies. Left panel, representative experiment is shown. Right panel, quantification of the results is shown. The results are presented as fold induction compared to the control untreated cells (n = 3; means ± S.D; *p<0.05). (B) Naïve or Bcl-2-GFP stably expressing LNCaP cells were treated with 100 ng/ml NRG for the indicated time periods. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3 and anti-Bcl-2 antibodies. Left panel, representative blot is shown. Right panel, quantification of the results is presented as fold induction compared to the control untreated cells (n = 3; means ± S.D; *p<0.05). (C) Naïve and Bcl-2-GFP stably expressing LNCaP cells were tested for cell viability using the methylene blue staining assay. Cells were treated with 100 ng/ml NRG and the methylene blue assay was performed 60 h later. Results are presented as % of control, and are the mean ± S.D of 4–6 determinations (**p<0.0001). This experiment was repeated three times with similar results.

Mentions: Beclin 1, a component of the class-III PI3K complex, is a major known regulator of autophagy [41], [42]. To determine the involvement of this pathway in NRG-induced autophagy, LNCaP cells were transfected with sh-Beclin 1 or control scrambled sh-RNA expression vectors. As shown in Figure 7A, NRG treatment enhanced autophagy in the control cells. However in sh-Beclin 1 transfected cells, NRG-mediated autophagy was reduced compared to the control cells. These results indicate that Beclin 1 protein is involved in NRG mediated autophagy in LNCaP cells.


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 Beclin-1 dependent.(A) LNCaP cells were transfected with Beclin 1 sh-RNA (3 µg) or control scrambled sh-RNA (3 µg) and incubated in normal medium for 72 h. The cells were then treated with 100 ng/ml NRG for additional 24 h. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3 and anti-Beclin 1 antibodies. Left panel, representative experiment is shown. Right panel, quantification of the results is shown. The results are presented as fold induction compared to the control untreated cells (n = 3; means ± S.D; *p<0.05). (B) Naïve or Bcl-2-GFP stably expressing LNCaP cells were treated with 100 ng/ml NRG for the indicated time periods. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3 and anti-Bcl-2 antibodies. Left panel, representative blot is shown. Right panel, quantification of the results is presented as fold induction compared to the control untreated cells (n = 3; means ± S.D; *p<0.05). (C) Naïve and Bcl-2-GFP stably expressing LNCaP cells were tested for cell viability using the methylene blue staining assay. Cells were treated with 100 ng/ml NRG and the methylene blue assay was performed 60 h later. Results are presented as % of control, and are the mean ± S.D of 4–6 determinations (**p<0.0001). This experiment was repeated three times with similar results.
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Related In: Results  -  Collection

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pone-0036828-g007: NRG-induced autophagy is Beclin-1 dependent.(A) LNCaP cells were transfected with Beclin 1 sh-RNA (3 µg) or control scrambled sh-RNA (3 µg) and incubated in normal medium for 72 h. The cells were then treated with 100 ng/ml NRG for additional 24 h. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3 and anti-Beclin 1 antibodies. Left panel, representative experiment is shown. Right panel, quantification of the results is shown. The results are presented as fold induction compared to the control untreated cells (n = 3; means ± S.D; *p<0.05). (B) Naïve or Bcl-2-GFP stably expressing LNCaP cells were treated with 100 ng/ml NRG for the indicated time periods. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3 and anti-Bcl-2 antibodies. Left panel, representative blot is shown. Right panel, quantification of the results is presented as fold induction compared to the control untreated cells (n = 3; means ± S.D; *p<0.05). (C) Naïve and Bcl-2-GFP stably expressing LNCaP cells were tested for cell viability using the methylene blue staining assay. Cells were treated with 100 ng/ml NRG and the methylene blue assay was performed 60 h later. Results are presented as % of control, and are the mean ± S.D of 4–6 determinations (**p<0.0001). This experiment was repeated three times with similar results.
Mentions: Beclin 1, a component of the class-III PI3K complex, is a major known regulator of autophagy [41], [42]. To determine the involvement of this pathway in NRG-induced autophagy, LNCaP cells were transfected with sh-Beclin 1 or control scrambled sh-RNA expression vectors. As shown in Figure 7A, NRG treatment enhanced autophagy in the control cells. However in sh-Beclin 1 transfected cells, NRG-mediated autophagy was reduced compared to the control cells. These results indicate that Beclin 1 protein is involved in NRG mediated autophagy in LNCaP cells.

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