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Expression and regulation of prostate apoptosis response-4 (Par-4) in human glioma stem cells in drug-induced apoptosis.

Jagtap JC, Dawood P, Shah RD, Chandrika G, Natesh K, Shiras A, Hegde AS, Ranade D, Shastry P - PLoS ONE (2014)

Bottom Line: We also demonstrate that the mechanism involves break down of mitochondrial membrane potential, down regulation of Bcl-2 and reduced activation of Akt and ERK 42/44.Secretory Par-4 and GRP-78 were significantly expressed in HNGC-2 cells on exposure to TAM and specific antibodies to these molecules inhibited cell death suggesting that extrinsic Par-4 is important in TAM-induced apoptosis.Interestingly, TAM decreased the expression of neural stem cell markers--Nestin, Bmi1, Vimentin, Sox2, and Musashi in HNGC-2 cell line and G1 cells implicating its potential as a stemness inhibiting drug.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Cell Science (NCCS), Pune, India.

ABSTRACT
Gliomas are the most common and aggressive of brain tumors in adults. Cancer stem cells (CSC) contribute to chemoresistance in many solid tumors including gliomas. The function of prostate apoptosis response-4 (Par-4) as a pro-apoptotic protein is well documented in many cancers; however, its role in CSC remains obscure. In this study, we aimed to explore the role of Par-4 in drug-induced cytotoxicity using human glioma stem cell line--HNGC-2 and primary culture (G1) derived from high grade glioma. We show that among the panel of drugs- lomustine, carmustine, UCN-01, oxaliplatin, temozolomide and tamoxifen (TAM) screened, only TAM induced cell death and up-regulated Par-4 levels significantly. TAM-induced apoptosis was confirmed by PARP cleavage, Annexin V and propidium iodide staining and caspase-3 activity. Knock down of Par-4 by siRNA inhibited cell death by TAM, suggesting the role of Par-4 in induction of apoptosis. We also demonstrate that the mechanism involves break down of mitochondrial membrane potential, down regulation of Bcl-2 and reduced activation of Akt and ERK 42/44. Secretory Par-4 and GRP-78 were significantly expressed in HNGC-2 cells on exposure to TAM and specific antibodies to these molecules inhibited cell death suggesting that extrinsic Par-4 is important in TAM-induced apoptosis. Interestingly, TAM decreased the expression of neural stem cell markers--Nestin, Bmi1, Vimentin, Sox2, and Musashi in HNGC-2 cell line and G1 cells implicating its potential as a stemness inhibiting drug. Based on these data and our findings that enhanced levels of Par-4 sensitize the resistant glioma stem cells to drug-induced apoptosis, we propose that Par-4 may be explored for evaluating anti-tumor agents in CSC.

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Tamoxifen induced Par-4 regulates Akt and ERK signaling in HNGC-2 cells.HNGC-2 cells treated with TAM (10 µg/mL) for increasing time periods were analyzed for the expression pattern of (A) pAkt (Ser473),total Akt and (B) pERK (42/44) and total ERK by western blotting. GAPDH was used as loading control. (C) HNGC-2 cells were transfected with control siRNA and Par-4 siRNA (100 nM) and analyzed for expression of Par-4 by immunofluorescence. (D) Transfected control and Par-4 silenced cells were analyzed for phosphoAkt (Ser473) total Akt and (E) phosphoERK and total ERK signaling by western blotting. Fold change is shown by bars, mean ± SD of three independent experiments. *p<0.05, control siRNA vs. Par-4 siRNA cells.
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pone-0088505-g006: Tamoxifen induced Par-4 regulates Akt and ERK signaling in HNGC-2 cells.HNGC-2 cells treated with TAM (10 µg/mL) for increasing time periods were analyzed for the expression pattern of (A) pAkt (Ser473),total Akt and (B) pERK (42/44) and total ERK by western blotting. GAPDH was used as loading control. (C) HNGC-2 cells were transfected with control siRNA and Par-4 siRNA (100 nM) and analyzed for expression of Par-4 by immunofluorescence. (D) Transfected control and Par-4 silenced cells were analyzed for phosphoAkt (Ser473) total Akt and (E) phosphoERK and total ERK signaling by western blotting. Fold change is shown by bars, mean ± SD of three independent experiments. *p<0.05, control siRNA vs. Par-4 siRNA cells.

Mentions: We next studied the effect of TAM in regulation of signaling pathways involving Akt and ERK 42/44, in HNGC-2 cells. The western blot analysis revealed that TAM significantly decreased the activation of Akt (ser473) and ERK42/44 in a time dependent manner suggesting their involvement in tamoxifen-induced apoptosis (Fig. 6A and B). In this regard, as TAM increased the expression of Par-4, it was of interest to examine whether Par-4 modulated the expression of Akt and ERK42/44. For this purpose, HNGC-2 cells were transfected with Par-4 specific siRNA or non-target siRNA and analysed for phosphorylated Akt and ERK42/44. As shown in Fig. 6D, the expression of phosphorylated Akt (ser473) was significantly (1.53 fold) enhanced in Par-4 knock down cells but loss of expression of Par-4 had no effect on the levels of phosphorylated and total Erk42/44 (Fig. 6E). These data suggested a role for Par-4 in Akt-mediated signalling but not in activation of ERK. However, based on the kinetics of expression of Par-4, phosphorylated Akt (Ser 473) and ERK42/44, it may be concluded that cellular mechanisms other than those involved in the downstream effects of PAR-4 may also contribute to Tamoxifen-induced PAR-4 activation.


Expression and regulation of prostate apoptosis response-4 (Par-4) in human glioma stem cells in drug-induced apoptosis.

Jagtap JC, Dawood P, Shah RD, Chandrika G, Natesh K, Shiras A, Hegde AS, Ranade D, Shastry P - PLoS ONE (2014)

Tamoxifen induced Par-4 regulates Akt and ERK signaling in HNGC-2 cells.HNGC-2 cells treated with TAM (10 µg/mL) for increasing time periods were analyzed for the expression pattern of (A) pAkt (Ser473),total Akt and (B) pERK (42/44) and total ERK by western blotting. GAPDH was used as loading control. (C) HNGC-2 cells were transfected with control siRNA and Par-4 siRNA (100 nM) and analyzed for expression of Par-4 by immunofluorescence. (D) Transfected control and Par-4 silenced cells were analyzed for phosphoAkt (Ser473) total Akt and (E) phosphoERK and total ERK signaling by western blotting. Fold change is shown by bars, mean ± SD of three independent experiments. *p<0.05, control siRNA vs. Par-4 siRNA cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921173&req=5

pone-0088505-g006: Tamoxifen induced Par-4 regulates Akt and ERK signaling in HNGC-2 cells.HNGC-2 cells treated with TAM (10 µg/mL) for increasing time periods were analyzed for the expression pattern of (A) pAkt (Ser473),total Akt and (B) pERK (42/44) and total ERK by western blotting. GAPDH was used as loading control. (C) HNGC-2 cells were transfected with control siRNA and Par-4 siRNA (100 nM) and analyzed for expression of Par-4 by immunofluorescence. (D) Transfected control and Par-4 silenced cells were analyzed for phosphoAkt (Ser473) total Akt and (E) phosphoERK and total ERK signaling by western blotting. Fold change is shown by bars, mean ± SD of three independent experiments. *p<0.05, control siRNA vs. Par-4 siRNA cells.
Mentions: We next studied the effect of TAM in regulation of signaling pathways involving Akt and ERK 42/44, in HNGC-2 cells. The western blot analysis revealed that TAM significantly decreased the activation of Akt (ser473) and ERK42/44 in a time dependent manner suggesting their involvement in tamoxifen-induced apoptosis (Fig. 6A and B). In this regard, as TAM increased the expression of Par-4, it was of interest to examine whether Par-4 modulated the expression of Akt and ERK42/44. For this purpose, HNGC-2 cells were transfected with Par-4 specific siRNA or non-target siRNA and analysed for phosphorylated Akt and ERK42/44. As shown in Fig. 6D, the expression of phosphorylated Akt (ser473) was significantly (1.53 fold) enhanced in Par-4 knock down cells but loss of expression of Par-4 had no effect on the levels of phosphorylated and total Erk42/44 (Fig. 6E). These data suggested a role for Par-4 in Akt-mediated signalling but not in activation of ERK. However, based on the kinetics of expression of Par-4, phosphorylated Akt (Ser 473) and ERK42/44, it may be concluded that cellular mechanisms other than those involved in the downstream effects of PAR-4 may also contribute to Tamoxifen-induced PAR-4 activation.

Bottom Line: We also demonstrate that the mechanism involves break down of mitochondrial membrane potential, down regulation of Bcl-2 and reduced activation of Akt and ERK 42/44.Secretory Par-4 and GRP-78 were significantly expressed in HNGC-2 cells on exposure to TAM and specific antibodies to these molecules inhibited cell death suggesting that extrinsic Par-4 is important in TAM-induced apoptosis.Interestingly, TAM decreased the expression of neural stem cell markers--Nestin, Bmi1, Vimentin, Sox2, and Musashi in HNGC-2 cell line and G1 cells implicating its potential as a stemness inhibiting drug.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Cell Science (NCCS), Pune, India.

ABSTRACT
Gliomas are the most common and aggressive of brain tumors in adults. Cancer stem cells (CSC) contribute to chemoresistance in many solid tumors including gliomas. The function of prostate apoptosis response-4 (Par-4) as a pro-apoptotic protein is well documented in many cancers; however, its role in CSC remains obscure. In this study, we aimed to explore the role of Par-4 in drug-induced cytotoxicity using human glioma stem cell line--HNGC-2 and primary culture (G1) derived from high grade glioma. We show that among the panel of drugs- lomustine, carmustine, UCN-01, oxaliplatin, temozolomide and tamoxifen (TAM) screened, only TAM induced cell death and up-regulated Par-4 levels significantly. TAM-induced apoptosis was confirmed by PARP cleavage, Annexin V and propidium iodide staining and caspase-3 activity. Knock down of Par-4 by siRNA inhibited cell death by TAM, suggesting the role of Par-4 in induction of apoptosis. We also demonstrate that the mechanism involves break down of mitochondrial membrane potential, down regulation of Bcl-2 and reduced activation of Akt and ERK 42/44. Secretory Par-4 and GRP-78 were significantly expressed in HNGC-2 cells on exposure to TAM and specific antibodies to these molecules inhibited cell death suggesting that extrinsic Par-4 is important in TAM-induced apoptosis. Interestingly, TAM decreased the expression of neural stem cell markers--Nestin, Bmi1, Vimentin, Sox2, and Musashi in HNGC-2 cell line and G1 cells implicating its potential as a stemness inhibiting drug. Based on these data and our findings that enhanced levels of Par-4 sensitize the resistant glioma stem cells to drug-induced apoptosis, we propose that Par-4 may be explored for evaluating anti-tumor agents in CSC.

Show MeSH
Related in: MedlinePlus