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Secretory prostate apoptosis response (Par)-4 sensitizes multicellular spheroids (MCS) of glioblastoma multiforme cells to tamoxifen-induced cell death.

Jagtap JC, Parveen D, Shah RD, Desai A, Bhosale D, Chugh A, Ranade D, Karnik S, Khedkar B, Mathur A, Natesh K, Chandrika G, Shastry P - FEBS Open Bio (2014)

Bottom Line: TAM effectively induced secretory Par-4 in conditioned medium (CM) of cells cultured as monolayer but not in MCS.Also TAM reduced the expression of Akt and PKCζ in GBM cells cultured as monolayer but not in MCS.Importantly, combination of TAM with inhibitors to PI3K inhibitor (LY294002) or PKCζ resulted in secretion of Par-4 and cell death in MCS.

View Article: PubMed Central - PubMed

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

ABSTRACT
Glioblastoma multiforme (GBM) is the most malignant form of brain tumor and is associated with resistance to conventional therapy and poor patient survival. Prostate apoptosis response (Par)-4, a tumor suppressor, is expressed as both an intracellular and secretory/extracellular protein. Though secretory Par-4 induces apoptosis in cancer cells, its potential in drug-resistant tumors remains to be fully explored. Multicellular spheroids (MCS) of cancer cells often acquire multi-drug resistance and serve as ideal experimental models. We investigated the role of Par-4 in Tamoxifen (TAM)-induced cell death in MCS of human cell lines and primary cultures of GBM tumors. TCGA and REMBRANT data analysis revealed that low levels of Par-4 correlated with low survival period (21.85 ± 19.30 days) in GBM but not in astrocytomas (59.13 ± 47.26 days) and oligodendrogliomas (58.04 ± 59.80 days) suggesting low PAWR expression as a predictive risk factor in GBM. Consistently, MCS of human cell lines and primary cultures displayed low Par-4 expression, high level of chemo-resistance genes and were resistant to TAM-induced cytotoxicity. In monolayer cells, TAM-induced cytotoxicity was associated with enhanced expression of Par-4 and was alleviated by silencing of Par-4 using specific siRNA. TAM effectively induced secretory Par-4 in conditioned medium (CM) of cells cultured as monolayer but not in MCS. Moreover, MCS were rendered sensitive to TAM-induced cell death by exposure to conditioned medium (CM)-containing Par-4 (derived from TAM-treated monolayer cells). Also TAM reduced the expression of Akt and PKCζ in GBM cells cultured as monolayer but not in MCS. Importantly, combination of TAM with inhibitors to PI3K inhibitor (LY294002) or PKCζ resulted in secretion of Par-4 and cell death in MCS. Since membrane GRP78 is overexpressed in most cancer cells but not normal cells, and secretory Par-4 induces apoptosis by binding to membrane GRP78, secretory Par-4 is an attractive candidate for potentially overcoming therapy-resistance not only in malignant glioma but in broad spectrum of cancers.

No MeSH data available.


Related in: MedlinePlus

TAM induces secretory Par-4 in ML but not MCS. (A) G1 cells cultured as ML and MCS were treated with TAM for 24 h and supernatants were analyzed for secretory Par-4 by Western blotting. The blots were stained with Ponceau and BSA was used as loading control (lower panel). (B) MCS of G1 were treated with combination of TAM and secretory Par-4 derived from HNGC-2 cells exposed to TAM (described in Section 2). Supernatant of untreated HNGC-2 cells was used as control. Cell viability was assessed after 24 h by MTT assay. (C) MCS of G1 cells were exposed to supernatant containing Par-4 that was pre incubated for 30 min with Par-4 antibody and cell viability was assessed by MTT assay. Antibody to p35 and species specific IgG were used as internal control. (D) Expression of GRP78 in ML and MCS of TAM treated G1 cells was assessed by Western blotting. GAPDH was used as loading control. ∗p < 0.05 is difference in viability between cells-treated with TAM in combination with conditioned medium containing Par-4 vs control supernatant (Fig. B) and in the presence of Par-4 antibody vs control antibody(Fig. C).
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f0040: TAM induces secretory Par-4 in ML but not MCS. (A) G1 cells cultured as ML and MCS were treated with TAM for 24 h and supernatants were analyzed for secretory Par-4 by Western blotting. The blots were stained with Ponceau and BSA was used as loading control (lower panel). (B) MCS of G1 were treated with combination of TAM and secretory Par-4 derived from HNGC-2 cells exposed to TAM (described in Section 2). Supernatant of untreated HNGC-2 cells was used as control. Cell viability was assessed after 24 h by MTT assay. (C) MCS of G1 cells were exposed to supernatant containing Par-4 that was pre incubated for 30 min with Par-4 antibody and cell viability was assessed by MTT assay. Antibody to p35 and species specific IgG were used as internal control. (D) Expression of GRP78 in ML and MCS of TAM treated G1 cells was assessed by Western blotting. GAPDH was used as loading control. ∗p < 0.05 is difference in viability between cells-treated with TAM in combination with conditioned medium containing Par-4 vs control supernatant (Fig. B) and in the presence of Par-4 antibody vs control antibody(Fig. C).

Mentions: We have previously demonstrated the role of secretory Par-4 in TAM-induced apoptosis in glioma stem cell line-HNGC-2 [25]. To address the question to what might be the factors that contribute to sensitivity/resistance in monolayers and MCS, we chose to analyze the conditioned medium (CM) of G1 cells treated with TAM. Interestingly, significant level of Par-4 was detected in supernatants of cells cultured as monolayer but not in MCS (Fig. 8A). Based on these data, we speculated whether addition of exogenous Par-4 would render MCS sensitive to TAM-induced apoptosis. For this purpose, we treated MCS of G1 cells with TAM in the presence of CM derived from HNGC-2 cells treated with TAM and assessed for cell viability. We observed that MCS exposed to TAM showed reduced viability in the presence of conditioned medium (CM) containing secretory Par-4 but not control CM. Also, treatment with CM-containing secretory Par-4, alone did not affect cell viability (Fig. 8B).


Secretory prostate apoptosis response (Par)-4 sensitizes multicellular spheroids (MCS) of glioblastoma multiforme cells to tamoxifen-induced cell death.

Jagtap JC, Parveen D, Shah RD, Desai A, Bhosale D, Chugh A, Ranade D, Karnik S, Khedkar B, Mathur A, Natesh K, Chandrika G, Shastry P - FEBS Open Bio (2014)

TAM induces secretory Par-4 in ML but not MCS. (A) G1 cells cultured as ML and MCS were treated with TAM for 24 h and supernatants were analyzed for secretory Par-4 by Western blotting. The blots were stained with Ponceau and BSA was used as loading control (lower panel). (B) MCS of G1 were treated with combination of TAM and secretory Par-4 derived from HNGC-2 cells exposed to TAM (described in Section 2). Supernatant of untreated HNGC-2 cells was used as control. Cell viability was assessed after 24 h by MTT assay. (C) MCS of G1 cells were exposed to supernatant containing Par-4 that was pre incubated for 30 min with Par-4 antibody and cell viability was assessed by MTT assay. Antibody to p35 and species specific IgG were used as internal control. (D) Expression of GRP78 in ML and MCS of TAM treated G1 cells was assessed by Western blotting. GAPDH was used as loading control. ∗p < 0.05 is difference in viability between cells-treated with TAM in combination with conditioned medium containing Par-4 vs control supernatant (Fig. B) and in the presence of Par-4 antibody vs control antibody(Fig. C).
© Copyright Policy - CC BY
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4309838&req=5

f0040: TAM induces secretory Par-4 in ML but not MCS. (A) G1 cells cultured as ML and MCS were treated with TAM for 24 h and supernatants were analyzed for secretory Par-4 by Western blotting. The blots were stained with Ponceau and BSA was used as loading control (lower panel). (B) MCS of G1 were treated with combination of TAM and secretory Par-4 derived from HNGC-2 cells exposed to TAM (described in Section 2). Supernatant of untreated HNGC-2 cells was used as control. Cell viability was assessed after 24 h by MTT assay. (C) MCS of G1 cells were exposed to supernatant containing Par-4 that was pre incubated for 30 min with Par-4 antibody and cell viability was assessed by MTT assay. Antibody to p35 and species specific IgG were used as internal control. (D) Expression of GRP78 in ML and MCS of TAM treated G1 cells was assessed by Western blotting. GAPDH was used as loading control. ∗p < 0.05 is difference in viability between cells-treated with TAM in combination with conditioned medium containing Par-4 vs control supernatant (Fig. B) and in the presence of Par-4 antibody vs control antibody(Fig. C).
Mentions: We have previously demonstrated the role of secretory Par-4 in TAM-induced apoptosis in glioma stem cell line-HNGC-2 [25]. To address the question to what might be the factors that contribute to sensitivity/resistance in monolayers and MCS, we chose to analyze the conditioned medium (CM) of G1 cells treated with TAM. Interestingly, significant level of Par-4 was detected in supernatants of cells cultured as monolayer but not in MCS (Fig. 8A). Based on these data, we speculated whether addition of exogenous Par-4 would render MCS sensitive to TAM-induced apoptosis. For this purpose, we treated MCS of G1 cells with TAM in the presence of CM derived from HNGC-2 cells treated with TAM and assessed for cell viability. We observed that MCS exposed to TAM showed reduced viability in the presence of conditioned medium (CM) containing secretory Par-4 but not control CM. Also, treatment with CM-containing secretory Par-4, alone did not affect cell viability (Fig. 8B).

Bottom Line: TAM effectively induced secretory Par-4 in conditioned medium (CM) of cells cultured as monolayer but not in MCS.Also TAM reduced the expression of Akt and PKCζ in GBM cells cultured as monolayer but not in MCS.Importantly, combination of TAM with inhibitors to PI3K inhibitor (LY294002) or PKCζ resulted in secretion of Par-4 and cell death in MCS.

View Article: PubMed Central - PubMed

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

ABSTRACT
Glioblastoma multiforme (GBM) is the most malignant form of brain tumor and is associated with resistance to conventional therapy and poor patient survival. Prostate apoptosis response (Par)-4, a tumor suppressor, is expressed as both an intracellular and secretory/extracellular protein. Though secretory Par-4 induces apoptosis in cancer cells, its potential in drug-resistant tumors remains to be fully explored. Multicellular spheroids (MCS) of cancer cells often acquire multi-drug resistance and serve as ideal experimental models. We investigated the role of Par-4 in Tamoxifen (TAM)-induced cell death in MCS of human cell lines and primary cultures of GBM tumors. TCGA and REMBRANT data analysis revealed that low levels of Par-4 correlated with low survival period (21.85 ± 19.30 days) in GBM but not in astrocytomas (59.13 ± 47.26 days) and oligodendrogliomas (58.04 ± 59.80 days) suggesting low PAWR expression as a predictive risk factor in GBM. Consistently, MCS of human cell lines and primary cultures displayed low Par-4 expression, high level of chemo-resistance genes and were resistant to TAM-induced cytotoxicity. In monolayer cells, TAM-induced cytotoxicity was associated with enhanced expression of Par-4 and was alleviated by silencing of Par-4 using specific siRNA. TAM effectively induced secretory Par-4 in conditioned medium (CM) of cells cultured as monolayer but not in MCS. Moreover, MCS were rendered sensitive to TAM-induced cell death by exposure to conditioned medium (CM)-containing Par-4 (derived from TAM-treated monolayer cells). Also TAM reduced the expression of Akt and PKCζ in GBM cells cultured as monolayer but not in MCS. Importantly, combination of TAM with inhibitors to PI3K inhibitor (LY294002) or PKCζ resulted in secretion of Par-4 and cell death in MCS. Since membrane GRP78 is overexpressed in most cancer cells but not normal cells, and secretory Par-4 induces apoptosis by binding to membrane GRP78, secretory Par-4 is an attractive candidate for potentially overcoming therapy-resistance not only in malignant glioma but in broad spectrum of cancers.

No MeSH data available.


Related in: MedlinePlus