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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 induces cell death by apoptosis.(A) HNGC-2 cells were exposed to lomustine (Lom), carmustine (Carm), UCN-01, oxaliplatin (Ox), tamoxifen (TAM) and temozolomide (TMZ) for 12 h and stained by Annexin V (FL-1) and Propidium Iodide (FL-2) to perform flowcytometry analysis. The graph represents percent apoptotic population (Annexin V and PI positive). The bars represent mean± SE of three independent experiments. *p<0.05, treated vs. vehicle control (V.C). (B)Western blot depicts total and cleaved PARP in HNGC-2 cells treated with panel of drugs for 24 h, GAPDH was used as loading control. (C) HNGC-2 cells were treated with TAM for 24 h in the presence or absence of caspase-3 inhibitor (ZVAD-FMK), stained with FITC-DEVD-FMK and analyzed for caspase-3 activity. The values in the graphs are percent positive cells for caspase-3 activity. Histogram is representative data of two similar experiments.
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pone-0088505-g002: Tamoxifen induces cell death by apoptosis.(A) HNGC-2 cells were exposed to lomustine (Lom), carmustine (Carm), UCN-01, oxaliplatin (Ox), tamoxifen (TAM) and temozolomide (TMZ) for 12 h and stained by Annexin V (FL-1) and Propidium Iodide (FL-2) to perform flowcytometry analysis. The graph represents percent apoptotic population (Annexin V and PI positive). The bars represent mean± SE of three independent experiments. *p<0.05, treated vs. vehicle control (V.C). (B)Western blot depicts total and cleaved PARP in HNGC-2 cells treated with panel of drugs for 24 h, GAPDH was used as loading control. (C) HNGC-2 cells were treated with TAM for 24 h in the presence or absence of caspase-3 inhibitor (ZVAD-FMK), stained with FITC-DEVD-FMK and analyzed for caspase-3 activity. The values in the graphs are percent positive cells for caspase-3 activity. Histogram is representative data of two similar experiments.

Mentions: To investigate whether the reduction in cell viability triggered by TAM was associated with apoptosis, dual staining with Annexin- V-FITC and propidium iodide was carried out. As shown in Fig. 2A, TAM induced significant apoptosis in comparison to other drugs used in the panel. Vehicle control cells showed 95% cell viability. For all further experiments untreated cell population was used as control. Western blotting analysis using antibody detecting total and cleaved PARP revealed that cell lysates from TAM-treated cells indicated increased band intensity corresponding to cleaved PARP. In the lysates of cells exposed to oxaliplatin a weak signal to cleaved PARP was observed (Fig. 2B). We further examined whether caspase-3 activity is involved in TAM induced-apoptosis using flowcytometric assay. We found that 61.28% of the cells treated with TAM for 24 h were positive for caspase-3 activity compared to 4.56% in untreated cells while caspase-3 inhibitor (ZVAD-FMK) reduced this population to 35.07% in TAM treated cells (Fig. 2C). These preliminary results suggested that HNGC-2 is sensitive to TAM-induced cell death that involved activation of caspase-3.


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 induces cell death by apoptosis.(A) HNGC-2 cells were exposed to lomustine (Lom), carmustine (Carm), UCN-01, oxaliplatin (Ox), tamoxifen (TAM) and temozolomide (TMZ) for 12 h and stained by Annexin V (FL-1) and Propidium Iodide (FL-2) to perform flowcytometry analysis. The graph represents percent apoptotic population (Annexin V and PI positive). The bars represent mean± SE of three independent experiments. *p<0.05, treated vs. vehicle control (V.C). (B)Western blot depicts total and cleaved PARP in HNGC-2 cells treated with panel of drugs for 24 h, GAPDH was used as loading control. (C) HNGC-2 cells were treated with TAM for 24 h in the presence or absence of caspase-3 inhibitor (ZVAD-FMK), stained with FITC-DEVD-FMK and analyzed for caspase-3 activity. The values in the graphs are percent positive cells for caspase-3 activity. Histogram is representative data of two similar experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0088505-g002: Tamoxifen induces cell death by apoptosis.(A) HNGC-2 cells were exposed to lomustine (Lom), carmustine (Carm), UCN-01, oxaliplatin (Ox), tamoxifen (TAM) and temozolomide (TMZ) for 12 h and stained by Annexin V (FL-1) and Propidium Iodide (FL-2) to perform flowcytometry analysis. The graph represents percent apoptotic population (Annexin V and PI positive). The bars represent mean± SE of three independent experiments. *p<0.05, treated vs. vehicle control (V.C). (B)Western blot depicts total and cleaved PARP in HNGC-2 cells treated with panel of drugs for 24 h, GAPDH was used as loading control. (C) HNGC-2 cells were treated with TAM for 24 h in the presence or absence of caspase-3 inhibitor (ZVAD-FMK), stained with FITC-DEVD-FMK and analyzed for caspase-3 activity. The values in the graphs are percent positive cells for caspase-3 activity. Histogram is representative data of two similar experiments.
Mentions: To investigate whether the reduction in cell viability triggered by TAM was associated with apoptosis, dual staining with Annexin- V-FITC and propidium iodide was carried out. As shown in Fig. 2A, TAM induced significant apoptosis in comparison to other drugs used in the panel. Vehicle control cells showed 95% cell viability. For all further experiments untreated cell population was used as control. Western blotting analysis using antibody detecting total and cleaved PARP revealed that cell lysates from TAM-treated cells indicated increased band intensity corresponding to cleaved PARP. In the lysates of cells exposed to oxaliplatin a weak signal to cleaved PARP was observed (Fig. 2B). We further examined whether caspase-3 activity is involved in TAM induced-apoptosis using flowcytometric assay. We found that 61.28% of the cells treated with TAM for 24 h were positive for caspase-3 activity compared to 4.56% in untreated cells while caspase-3 inhibitor (ZVAD-FMK) reduced this population to 35.07% in TAM treated cells (Fig. 2C). These preliminary results suggested that HNGC-2 is sensitive to TAM-induced cell death that involved activation of caspase-3.

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