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The anti-apoptotic BAG3 protein is expressed in lung carcinomas and regulates small cell lung carcinoma (SCLC) tumor growth.

Chiappetta G, Basile A, Barbieri A, Falco A, Rosati A, Festa M, Pasquinelli R, Califano D, Palma G, Costanzo R, Barcaroli D, Capunzo M, Franco R, Rocco G, Pascale M, Turco MC, De Laurenzi V, Arra C - Oncotarget (2014)

Bottom Line: Normal lung tissue did not express BAG3 while we detected the expression of BAG3 by immunohistochemistry in all the 13 squamous cell carcinomas, 13 adenocarcinomas and 4 large cell carcinomas.Treatment with bag3 siRNA-Ad significantly reduced tumor growth and improved animal survival.In conclusion we show that a subset of SCLCs over express BAG3 that exerts an anti-apoptotic effect resulting in resistance to chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Functional Genomic Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Giovanni Pascale", IRCCS, Naples, Italy. .

ABSTRACT
BAG3, member the HSP70 co-chaperones family, has been shown to play a relevant role in the survival, growth and invasiveness of different tumor types. In this study, we investigate the expression of BAG3 in 66 specimens from different lung tumors and the role of this protein in small cell lung cancer (SCLC) tumor growth. Normal lung tissue did not express BAG3 while we detected the expression of BAG3 by immunohistochemistry in all the 13 squamous cell carcinomas, 13 adenocarcinomas and 4 large cell carcinomas. Furthermore, we detected BAG3 expression in 22 of the 36 SCLCs analyzed. The role on SCLC cell survival was determined by down-regulating BAG3 levels in two human SCLC cell lines, i.e. H69 and H446, in vitro and measuring cisplatin induced apoptosis. Indeed down-regulation of BAG3 determines increased cell death and sensitizes cells to cisplatin treatment. The effect of BAG3 down-regulation on tumor growth was also investigated in an in vivo xenograft model by treating mice with an adenovirus expressing a specific bag3 siRNA. Treatment with bag3 siRNA-Ad significantly reduced tumor growth and improved animal survival. In conclusion we show that a subset of SCLCs over express BAG3 that exerts an anti-apoptotic effect resulting in resistance to chemotherapy.

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Analysis of BAG3 protein levels in human SCLC cell linesA) Total protein extracts obtained from DMS79, H526, H209, H69 and H446 cell lines were analyzed by western blot using an anti-BAG3 polyclonal antibody. Densitometry data of samples are expressed as fractions of BAG3/GAPDH. H69 cells (B) and H446 cells (C) were transfected with a bag3-specific (bag3 siRNA) (100 and 200 nM) or a NT siRNA (200 nM). After 72 hrs BAG3 protein levels were analyzed by western blot using an anti-BAG3 polyclonal antibody. Anti-GAPDH antibody was used as loading control.
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Figure 2: Analysis of BAG3 protein levels in human SCLC cell linesA) Total protein extracts obtained from DMS79, H526, H209, H69 and H446 cell lines were analyzed by western blot using an anti-BAG3 polyclonal antibody. Densitometry data of samples are expressed as fractions of BAG3/GAPDH. H69 cells (B) and H446 cells (C) were transfected with a bag3-specific (bag3 siRNA) (100 and 200 nM) or a NT siRNA (200 nM). After 72 hrs BAG3 protein levels were analyzed by western blot using an anti-BAG3 polyclonal antibody. Anti-GAPDH antibody was used as loading control.

Mentions: We then investigated the possibility that in BAG3 positive SCLCs this protein played a pro-survival role as reported for other tumor types [3, 4]. To this end we first evaluated BAG3 expression by western blot in five human SCLC cell lines. As shown in Fig. 2A, H69 and H446 cell lines displayed the highest BAG3 protein levels and were chosen for the subsequent experiments to test if BAG3 silencing sensitized cells to cisplatin treatment. Both cell lines were transfected with a bag3- specific small interfering (si) RNA or with a non targeted (NT) siRNA. As shown in figures 2B and C transfection with 200 nM of Bag3 specific siRNA resulted in silencing of BAG3 in both cell lines. As shown in figure 3B and D silencing of BAG3 results in increased response to cisplatin with an increase in both cell lines of almost 40% of apoptosis after 48 hours of treatment with a 100 μM cisplatin. Furthermore, BAG3 silencing in H446 results also a significant increase of basal apoptosis (Fig. 3D).


The anti-apoptotic BAG3 protein is expressed in lung carcinomas and regulates small cell lung carcinoma (SCLC) tumor growth.

Chiappetta G, Basile A, Barbieri A, Falco A, Rosati A, Festa M, Pasquinelli R, Califano D, Palma G, Costanzo R, Barcaroli D, Capunzo M, Franco R, Rocco G, Pascale M, Turco MC, De Laurenzi V, Arra C - Oncotarget (2014)

Analysis of BAG3 protein levels in human SCLC cell linesA) Total protein extracts obtained from DMS79, H526, H209, H69 and H446 cell lines were analyzed by western blot using an anti-BAG3 polyclonal antibody. Densitometry data of samples are expressed as fractions of BAG3/GAPDH. H69 cells (B) and H446 cells (C) were transfected with a bag3-specific (bag3 siRNA) (100 and 200 nM) or a NT siRNA (200 nM). After 72 hrs BAG3 protein levels were analyzed by western blot using an anti-BAG3 polyclonal antibody. Anti-GAPDH antibody was used as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196167&req=5

Figure 2: Analysis of BAG3 protein levels in human SCLC cell linesA) Total protein extracts obtained from DMS79, H526, H209, H69 and H446 cell lines were analyzed by western blot using an anti-BAG3 polyclonal antibody. Densitometry data of samples are expressed as fractions of BAG3/GAPDH. H69 cells (B) and H446 cells (C) were transfected with a bag3-specific (bag3 siRNA) (100 and 200 nM) or a NT siRNA (200 nM). After 72 hrs BAG3 protein levels were analyzed by western blot using an anti-BAG3 polyclonal antibody. Anti-GAPDH antibody was used as loading control.
Mentions: We then investigated the possibility that in BAG3 positive SCLCs this protein played a pro-survival role as reported for other tumor types [3, 4]. To this end we first evaluated BAG3 expression by western blot in five human SCLC cell lines. As shown in Fig. 2A, H69 and H446 cell lines displayed the highest BAG3 protein levels and were chosen for the subsequent experiments to test if BAG3 silencing sensitized cells to cisplatin treatment. Both cell lines were transfected with a bag3- specific small interfering (si) RNA or with a non targeted (NT) siRNA. As shown in figures 2B and C transfection with 200 nM of Bag3 specific siRNA resulted in silencing of BAG3 in both cell lines. As shown in figure 3B and D silencing of BAG3 results in increased response to cisplatin with an increase in both cell lines of almost 40% of apoptosis after 48 hours of treatment with a 100 μM cisplatin. Furthermore, BAG3 silencing in H446 results also a significant increase of basal apoptosis (Fig. 3D).

Bottom Line: Normal lung tissue did not express BAG3 while we detected the expression of BAG3 by immunohistochemistry in all the 13 squamous cell carcinomas, 13 adenocarcinomas and 4 large cell carcinomas.Treatment with bag3 siRNA-Ad significantly reduced tumor growth and improved animal survival.In conclusion we show that a subset of SCLCs over express BAG3 that exerts an anti-apoptotic effect resulting in resistance to chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Functional Genomic Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Giovanni Pascale", IRCCS, Naples, Italy. .

ABSTRACT
BAG3, member the HSP70 co-chaperones family, has been shown to play a relevant role in the survival, growth and invasiveness of different tumor types. In this study, we investigate the expression of BAG3 in 66 specimens from different lung tumors and the role of this protein in small cell lung cancer (SCLC) tumor growth. Normal lung tissue did not express BAG3 while we detected the expression of BAG3 by immunohistochemistry in all the 13 squamous cell carcinomas, 13 adenocarcinomas and 4 large cell carcinomas. Furthermore, we detected BAG3 expression in 22 of the 36 SCLCs analyzed. The role on SCLC cell survival was determined by down-regulating BAG3 levels in two human SCLC cell lines, i.e. H69 and H446, in vitro and measuring cisplatin induced apoptosis. Indeed down-regulation of BAG3 determines increased cell death and sensitizes cells to cisplatin treatment. The effect of BAG3 down-regulation on tumor growth was also investigated in an in vivo xenograft model by treating mice with an adenovirus expressing a specific bag3 siRNA. Treatment with bag3 siRNA-Ad significantly reduced tumor growth and improved animal survival. In conclusion we show that a subset of SCLCs over express BAG3 that exerts an anti-apoptotic effect resulting in resistance to chemotherapy.

Show MeSH
Related in: MedlinePlus