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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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SP600125 inhibits NRG-induced autophagy and cell death.(A) LNCaP cells were treated with 100 ng/ml NRG for 24 h with or without 20 µM SP600125. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3, anti-p62, anti-p-JNK and anti-JNK antibodies. Left panel, representative results. Right panel, densitometric analysis is presented as fold induction over the control untreated cells (n = 5; means ± S.D; *p<0.05). (B) LNCaP cells were treated with 100 ng/ml NRG in the presence or in the absence of 20 µM SP600125 for 60 h. The cells were stained with the fluorescent DNA dye bisbenzimide (Hoecsht 33258, 1 µg/ml) to assess the number of dying cells. Following staining, the cells were photographed using Olympus optical inverted phase-contrast microscope Model IX70 (20×magnification; scale bar, 50 micrometer). Left panel, representative images are shown. Right panel, percentage of dying cells was estimated by counting the number of Hoecsht-positive cells compared to the number total cells in each field (10–15 fields for each treatment, 100–200 cells per field). Results are presented as mean ± S.D (**p<0.0001). (C) LNCaP cells were tested for cell viability using the methylene blue staining assay. Cells were treated with 100 ng/ml NRG in the presence or in the absence of 20 µM SP600125. 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-g006: SP600125 inhibits NRG-induced autophagy and cell death.(A) LNCaP cells were treated with 100 ng/ml NRG for 24 h with or without 20 µM SP600125. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3, anti-p62, anti-p-JNK and anti-JNK antibodies. Left panel, representative results. Right panel, densitometric analysis is presented as fold induction over the control untreated cells (n = 5; means ± S.D; *p<0.05). (B) LNCaP cells were treated with 100 ng/ml NRG in the presence or in the absence of 20 µM SP600125 for 60 h. The cells were stained with the fluorescent DNA dye bisbenzimide (Hoecsht 33258, 1 µg/ml) to assess the number of dying cells. Following staining, the cells were photographed using Olympus optical inverted phase-contrast microscope Model IX70 (20×magnification; scale bar, 50 micrometer). Left panel, representative images are shown. Right panel, percentage of dying cells was estimated by counting the number of Hoecsht-positive cells compared to the number total cells in each field (10–15 fields for each treatment, 100–200 cells per field). Results are presented as mean ± S.D (**p<0.0001). (C) LNCaP cells were tested for cell viability using the methylene blue staining assay. Cells were treated with 100 ng/ml NRG in the presence or in the absence of 20 µM SP600125. 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: Because mTOR pathway is not involved in NRG-induced autophagy and cell death, we searched for other possible mediators. We chose to examine the involvement of JNK, since NRG induced JNK phosphorylation, which was inhibited by NAC. Therefore, we first examined the effect of JNK inhibitor SP600125 on NRG-induced autophagy (Figure 6A). As shown, in the presence of SP600125, NRG-induced autophagy was inhibited, as judged by the decreased LC3-II/LC3-I ratio. Yet, SP600125 treatment had no effect on p62 levels. Next, we examined the effect of JNK inhibitor on cell viability using Hoecsht dye exclusion and methylene blue staining assays (Fig. 6B and C, respectively). As shown, in the presence of JNK inhibitor, NRG-induced cell death was inhibited; indicating that JNK activation by NRG may be important for the induction of autophagy and cell death of 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)

SP600125 inhibits NRG-induced autophagy and cell death.(A) LNCaP cells were treated with 100 ng/ml NRG for 24 h with or without 20 µM SP600125. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3, anti-p62, anti-p-JNK and anti-JNK antibodies. Left panel, representative results. Right panel, densitometric analysis is presented as fold induction over the control untreated cells (n = 5; means ± S.D; *p<0.05). (B) LNCaP cells were treated with 100 ng/ml NRG in the presence or in the absence of 20 µM SP600125 for 60 h. The cells were stained with the fluorescent DNA dye bisbenzimide (Hoecsht 33258, 1 µg/ml) to assess the number of dying cells. Following staining, the cells were photographed using Olympus optical inverted phase-contrast microscope Model IX70 (20×magnification; scale bar, 50 micrometer). Left panel, representative images are shown. Right panel, percentage of dying cells was estimated by counting the number of Hoecsht-positive cells compared to the number total cells in each field (10–15 fields for each treatment, 100–200 cells per field). Results are presented as mean ± S.D (**p<0.0001). (C) LNCaP cells were tested for cell viability using the methylene blue staining assay. Cells were treated with 100 ng/ml NRG in the presence or in the absence of 20 µM SP600125. 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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getmorefigures.php?uid=PMC3351469&req=5

pone-0036828-g006: SP600125 inhibits NRG-induced autophagy and cell death.(A) LNCaP cells were treated with 100 ng/ml NRG for 24 h with or without 20 µM SP600125. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3, anti-p62, anti-p-JNK and anti-JNK antibodies. Left panel, representative results. Right panel, densitometric analysis is presented as fold induction over the control untreated cells (n = 5; means ± S.D; *p<0.05). (B) LNCaP cells were treated with 100 ng/ml NRG in the presence or in the absence of 20 µM SP600125 for 60 h. The cells were stained with the fluorescent DNA dye bisbenzimide (Hoecsht 33258, 1 µg/ml) to assess the number of dying cells. Following staining, the cells were photographed using Olympus optical inverted phase-contrast microscope Model IX70 (20×magnification; scale bar, 50 micrometer). Left panel, representative images are shown. Right panel, percentage of dying cells was estimated by counting the number of Hoecsht-positive cells compared to the number total cells in each field (10–15 fields for each treatment, 100–200 cells per field). Results are presented as mean ± S.D (**p<0.0001). (C) LNCaP cells were tested for cell viability using the methylene blue staining assay. Cells were treated with 100 ng/ml NRG in the presence or in the absence of 20 µM SP600125. 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: Because mTOR pathway is not involved in NRG-induced autophagy and cell death, we searched for other possible mediators. We chose to examine the involvement of JNK, since NRG induced JNK phosphorylation, which was inhibited by NAC. Therefore, we first examined the effect of JNK inhibitor SP600125 on NRG-induced autophagy (Figure 6A). As shown, in the presence of SP600125, NRG-induced autophagy was inhibited, as judged by the decreased LC3-II/LC3-I ratio. Yet, SP600125 treatment had no effect on p62 levels. Next, we examined the effect of JNK inhibitor on cell viability using Hoecsht dye exclusion and methylene blue staining assays (Fig. 6B and C, respectively). As shown, in the presence of JNK inhibitor, NRG-induced cell death was inhibited; indicating that JNK activation by NRG may be important for the induction of autophagy and cell death of 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