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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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Neuregulin induces autophagy in LNCaP cells.(A) LNCaP cells were treated with 100 ng/ml neuregulin (NRG) in the presence or in the absence of 10 mM 3-methyladenine (3-MA) for the indicated time period. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3 and anti-p62 antibodies. Upper panel, representative results. Lower panel, densitometric analysis is presented as fold induction over the control untreated cells (n = 3; means ± S.D; *p<0.05). (B) LNCaP cells stably expressing LC3-GFP were treated with 50 nM rapamycin for overnight or with 100 ng/ml NRG for 5 h. The cells were fixed with 4% paraformaldehyde and nuclei were stained with bisdenzimide (Hoecsht 33258). Following fixation and staining, the cells were photographed using Nikon optical fluorescence microscope Model TE-2000S (60×magnification). Upper panel, representative images. Lower panel, autophagy was quantified by counting the number LC3 dots per cell using the ImageJ software. The result shown is representative of two independent experiments. 70–80 cells were analyzed per treatment; data presented as mean ± S.D (*p<0.05).
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pone-0036828-g001: Neuregulin induces autophagy in LNCaP cells.(A) LNCaP cells were treated with 100 ng/ml neuregulin (NRG) in the presence or in the absence of 10 mM 3-methyladenine (3-MA) for the indicated time period. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3 and anti-p62 antibodies. Upper panel, representative results. Lower panel, densitometric analysis is presented as fold induction over the control untreated cells (n = 3; means ± S.D; *p<0.05). (B) LNCaP cells stably expressing LC3-GFP were treated with 50 nM rapamycin for overnight or with 100 ng/ml NRG for 5 h. The cells were fixed with 4% paraformaldehyde and nuclei were stained with bisdenzimide (Hoecsht 33258). Following fixation and staining, the cells were photographed using Nikon optical fluorescence microscope Model TE-2000S (60×magnification). Upper panel, representative images. Lower panel, autophagy was quantified by counting the number LC3 dots per cell using the ImageJ software. The result shown is representative of two independent experiments. 70–80 cells were analyzed per treatment; data presented as mean ± S.D (*p<0.05).

Mentions: LNCaP is an androgen-responsive prostate carcinoma cell line that expresses the ErbB receptors [29]. Previously, we demonstrated that NRG induces cell death that was inhibited by 3-MA, a PI3K inhibitor [29], and is caspase independent (Not shown and [29]). It was also demonstrated that NRG induces morphological changes in LNCaP cells that were inhibited by 3-MA (Video S1 and [29]). In the present study, we analyzed the effect of NRG on autophagy and cell death of LNCaP cells grown without androgen mimetic. In order to determine autophagy induction, we used LC3 protein as a marker. As shown in Figure 1A, LNCaP cells treated with NRG for 14 h and 24 h, exhibited enhanced conversion of LC3-I to LC3-II, which was inhibited by 3-MA, indicating that indeed NRG induces autophagy in LNCaP cells. To further demonstrate autophagy induction, we used LNCaP cells stably expressing GFP-LC3 expression vector. As shown in Figure 1B, NRG induced enhanced autophagosome formation as reflected by enhanced punctuated staining of GFP-LC3. As a positive control, cells were treated with rapamycin, which also induced autophagy in LNCaP cells (Figure 1B). Thus, LNCaP cells respond to NRG by increased autophagy induction. To further study autophagy induced by NRG, we examined the expression level of p62/SQSTM1. The p62/SQSTM1 protein binds LC3-II and is degraded by autophagy [30]. Surprisingly, NRG treatment did not enhance p62 degradation indicating that autophagy induced by NRG may be incomplete. As a control, cells were treated with Earle's balanced salt solution (EBSS) and the levels of LC3-II and p62 were determined by Immunoblot (Figure S1). As shown, EBSS induced LC3-II elevation and p62 degradation as expected. Of note, 3-MA treatment reduced LC3-II levels in NRG treated cells as well as p62 levels in the absence of NRG. The explanation for these results are yet unknown.


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)

Neuregulin induces autophagy in LNCaP cells.(A) LNCaP cells were treated with 100 ng/ml neuregulin (NRG) in the presence or in the absence of 10 mM 3-methyladenine (3-MA) for the indicated time period. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3 and anti-p62 antibodies. Upper panel, representative results. Lower panel, densitometric analysis is presented as fold induction over the control untreated cells (n = 3; means ± S.D; *p<0.05). (B) LNCaP cells stably expressing LC3-GFP were treated with 50 nM rapamycin for overnight or with 100 ng/ml NRG for 5 h. The cells were fixed with 4% paraformaldehyde and nuclei were stained with bisdenzimide (Hoecsht 33258). Following fixation and staining, the cells were photographed using Nikon optical fluorescence microscope Model TE-2000S (60×magnification). Upper panel, representative images. Lower panel, autophagy was quantified by counting the number LC3 dots per cell using the ImageJ software. The result shown is representative of two independent experiments. 70–80 cells were analyzed per treatment; data presented as mean ± S.D (*p<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351469&req=5

pone-0036828-g001: Neuregulin induces autophagy in LNCaP cells.(A) LNCaP cells were treated with 100 ng/ml neuregulin (NRG) in the presence or in the absence of 10 mM 3-methyladenine (3-MA) for the indicated time period. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3 and anti-p62 antibodies. Upper panel, representative results. Lower panel, densitometric analysis is presented as fold induction over the control untreated cells (n = 3; means ± S.D; *p<0.05). (B) LNCaP cells stably expressing LC3-GFP were treated with 50 nM rapamycin for overnight or with 100 ng/ml NRG for 5 h. The cells were fixed with 4% paraformaldehyde and nuclei were stained with bisdenzimide (Hoecsht 33258). Following fixation and staining, the cells were photographed using Nikon optical fluorescence microscope Model TE-2000S (60×magnification). Upper panel, representative images. Lower panel, autophagy was quantified by counting the number LC3 dots per cell using the ImageJ software. The result shown is representative of two independent experiments. 70–80 cells were analyzed per treatment; data presented as mean ± S.D (*p<0.05).
Mentions: LNCaP is an androgen-responsive prostate carcinoma cell line that expresses the ErbB receptors [29]. Previously, we demonstrated that NRG induces cell death that was inhibited by 3-MA, a PI3K inhibitor [29], and is caspase independent (Not shown and [29]). It was also demonstrated that NRG induces morphological changes in LNCaP cells that were inhibited by 3-MA (Video S1 and [29]). In the present study, we analyzed the effect of NRG on autophagy and cell death of LNCaP cells grown without androgen mimetic. In order to determine autophagy induction, we used LC3 protein as a marker. As shown in Figure 1A, LNCaP cells treated with NRG for 14 h and 24 h, exhibited enhanced conversion of LC3-I to LC3-II, which was inhibited by 3-MA, indicating that indeed NRG induces autophagy in LNCaP cells. To further demonstrate autophagy induction, we used LNCaP cells stably expressing GFP-LC3 expression vector. As shown in Figure 1B, NRG induced enhanced autophagosome formation as reflected by enhanced punctuated staining of GFP-LC3. As a positive control, cells were treated with rapamycin, which also induced autophagy in LNCaP cells (Figure 1B). Thus, LNCaP cells respond to NRG by increased autophagy induction. To further study autophagy induced by NRG, we examined the expression level of p62/SQSTM1. The p62/SQSTM1 protein binds LC3-II and is degraded by autophagy [30]. Surprisingly, NRG treatment did not enhance p62 degradation indicating that autophagy induced by NRG may be incomplete. As a control, cells were treated with Earle's balanced salt solution (EBSS) and the levels of LC3-II and p62 were determined by Immunoblot (Figure S1). As shown, EBSS induced LC3-II elevation and p62 degradation as expected. Of note, 3-MA treatment reduced LC3-II levels in NRG treated cells as well as p62 levels in the absence of NRG. The explanation for these results are yet unknown.

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