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Pterostilbene-isothiocyanate conjugate suppresses growth of prostate cancer cells irrespective of androgen receptor status.

Nikhil K, Sharan S, Chakraborty A, Roy P - PLoS ONE (2014)

Bottom Line: However, the effectiveness of these therapies is limited, thus necessitating the development of alternative approaches.The reduced proliferation of PC-3 as well as LNCaP cells by conjugate correlated with accumulation of cells in G2/M phase and induction of caspase dependent apoptosis.Moreover, anti-androgenic activity of the conjugate was mediated by decreased expression of AR and its co-activators (SRC-1, GRIP-1), thus interfering in their interactions with AR.

View Article: PubMed Central - PubMed

Affiliation: Molecular Endocrinology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India.

ABSTRACT
Chemotherapy and anti-hormonal therapies are the most common treatments for non-organ-confined prostate cancer (PCa). However, the effectiveness of these therapies is limited, thus necessitating the development of alternative approaches. The present study focused on analyzing the role of pterostilbene (PTER)-isothiocyanate (ITC) conjugate--a novel class of hybrid compound synthesized by appending an ITC moiety on PTER backbone--in regulating the functions of androgen receptor (AR), thereby causing apoptosis of PCa cells. The conjugate molecule caused 50% growth inhibition (IC50) at 40 ± 1.12 and 45 ± 1.50 μM in AR positive (LNCaP) and negative (PC-3) cells, respectively. The reduced proliferation of PC-3 as well as LNCaP cells by conjugate correlated with accumulation of cells in G2/M phase and induction of caspase dependent apoptosis. Both PI3K/Akt and MAPK/ERK pathways played an important and differential role in conjugate-induced apoptosis of these PCa cells. While the inhibitor of Akt (A6730) or Akt-specific small interference RNA (siRNA) greatly sensitized PC-3 cells to conjugate-induced apoptosis, on the contrary, apoptosis was accelerated by inhibition of ERK (by PD98059 or ERK siRNA) in case of LNCaP cells, both ultimately culminating in the expression of cleaved caspase-3 protein. Moreover, anti-androgenic activity of the conjugate was mediated by decreased expression of AR and its co-activators (SRC-1, GRIP-1), thus interfering in their interactions with AR. All these data suggests that conjugate-induced inhibition of cell proliferation and induction of apoptosis are partly mediated by the down regulation of AR, Akt, and ERK signaling. These observations provide a rationale for devising novel therapeutic approaches for treating PCa by using conjugate alone or in combination with other therapeutics.

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Conjugate inhibits androgen receptor transactivation and translocation in prostate cancer cells.Effect of conjugate on the transactivation of androgen receptor in presence/absence of 10 nM DHT in (A) androgen receptor positive LNCaP cells and (B) androgen receptor negative PC-3 cells. Luciferase activities are expressed as percentage of transactivation with respect to only 10 nM DHT treated group which is considered as 100%. *indicates statistically significant difference (p<0.05) with respect to DHT treated groups. (C) Effect of conjugate on the dynamics of nuclear translocation of androgen receptor as determined by green fluorescent protein (GFP)-androgen receptor construct in presence/absence of 10 nM DHT for 2 h.
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pone-0093335-g007: Conjugate inhibits androgen receptor transactivation and translocation in prostate cancer cells.Effect of conjugate on the transactivation of androgen receptor in presence/absence of 10 nM DHT in (A) androgen receptor positive LNCaP cells and (B) androgen receptor negative PC-3 cells. Luciferase activities are expressed as percentage of transactivation with respect to only 10 nM DHT treated group which is considered as 100%. *indicates statistically significant difference (p<0.05) with respect to DHT treated groups. (C) Effect of conjugate on the dynamics of nuclear translocation of androgen receptor as determined by green fluorescent protein (GFP)-androgen receptor construct in presence/absence of 10 nM DHT for 2 h.

Mentions: In order to check the anti-androgenic activity of conjugate, AR positive LNCaP cells were transiently transfected with pMMTV-neomycin-luciferase construct. Ligand-activated AR binds to androgen response element (ARE) and its functional activation was tested using a luciferase reporter gene linked to MMTV promoter having multiple repeats of AREs. The effect of DHT (10 nM) on the luciferase activity was expressed as 100% transactivation. The conjugate alone (without DHT) did not induce transcriptional activation at any of the concentrations tested (1–40 μM). However, as shown in Fig. 7A, the conjugate exhibited a dose-dependent anti-androgenic activity by inhibiting the androgen-induced transactivation by about 73% at the highest dose tested (40 μM) as compared to only DHT treatment. In order to confirm the anti-androgenic activity of conjugate further, similar transactivation assay was also performed in AR negative PC-3 cells co-transfected with pSG5-hAR and pMMTV-neomycin-luciferase constructs. As shown in Fig. 7B, consistent with the results described above for LNCaP cells, in this cell line (PC-3) also the DHT-mediated luciferase activity was reduced by 19, 31, 63, 70 and maximum to 80% in the presence of 5, 10, 20, 30 and 40 μM of conjugate, respectively.


Pterostilbene-isothiocyanate conjugate suppresses growth of prostate cancer cells irrespective of androgen receptor status.

Nikhil K, Sharan S, Chakraborty A, Roy P - PLoS ONE (2014)

Conjugate inhibits androgen receptor transactivation and translocation in prostate cancer cells.Effect of conjugate on the transactivation of androgen receptor in presence/absence of 10 nM DHT in (A) androgen receptor positive LNCaP cells and (B) androgen receptor negative PC-3 cells. Luciferase activities are expressed as percentage of transactivation with respect to only 10 nM DHT treated group which is considered as 100%. *indicates statistically significant difference (p<0.05) with respect to DHT treated groups. (C) Effect of conjugate on the dynamics of nuclear translocation of androgen receptor as determined by green fluorescent protein (GFP)-androgen receptor construct in presence/absence of 10 nM DHT for 2 h.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974779&req=5

pone-0093335-g007: Conjugate inhibits androgen receptor transactivation and translocation in prostate cancer cells.Effect of conjugate on the transactivation of androgen receptor in presence/absence of 10 nM DHT in (A) androgen receptor positive LNCaP cells and (B) androgen receptor negative PC-3 cells. Luciferase activities are expressed as percentage of transactivation with respect to only 10 nM DHT treated group which is considered as 100%. *indicates statistically significant difference (p<0.05) with respect to DHT treated groups. (C) Effect of conjugate on the dynamics of nuclear translocation of androgen receptor as determined by green fluorescent protein (GFP)-androgen receptor construct in presence/absence of 10 nM DHT for 2 h.
Mentions: In order to check the anti-androgenic activity of conjugate, AR positive LNCaP cells were transiently transfected with pMMTV-neomycin-luciferase construct. Ligand-activated AR binds to androgen response element (ARE) and its functional activation was tested using a luciferase reporter gene linked to MMTV promoter having multiple repeats of AREs. The effect of DHT (10 nM) on the luciferase activity was expressed as 100% transactivation. The conjugate alone (without DHT) did not induce transcriptional activation at any of the concentrations tested (1–40 μM). However, as shown in Fig. 7A, the conjugate exhibited a dose-dependent anti-androgenic activity by inhibiting the androgen-induced transactivation by about 73% at the highest dose tested (40 μM) as compared to only DHT treatment. In order to confirm the anti-androgenic activity of conjugate further, similar transactivation assay was also performed in AR negative PC-3 cells co-transfected with pSG5-hAR and pMMTV-neomycin-luciferase constructs. As shown in Fig. 7B, consistent with the results described above for LNCaP cells, in this cell line (PC-3) also the DHT-mediated luciferase activity was reduced by 19, 31, 63, 70 and maximum to 80% in the presence of 5, 10, 20, 30 and 40 μM of conjugate, respectively.

Bottom Line: However, the effectiveness of these therapies is limited, thus necessitating the development of alternative approaches.The reduced proliferation of PC-3 as well as LNCaP cells by conjugate correlated with accumulation of cells in G2/M phase and induction of caspase dependent apoptosis.Moreover, anti-androgenic activity of the conjugate was mediated by decreased expression of AR and its co-activators (SRC-1, GRIP-1), thus interfering in their interactions with AR.

View Article: PubMed Central - PubMed

Affiliation: Molecular Endocrinology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India.

ABSTRACT
Chemotherapy and anti-hormonal therapies are the most common treatments for non-organ-confined prostate cancer (PCa). However, the effectiveness of these therapies is limited, thus necessitating the development of alternative approaches. The present study focused on analyzing the role of pterostilbene (PTER)-isothiocyanate (ITC) conjugate--a novel class of hybrid compound synthesized by appending an ITC moiety on PTER backbone--in regulating the functions of androgen receptor (AR), thereby causing apoptosis of PCa cells. The conjugate molecule caused 50% growth inhibition (IC50) at 40 ± 1.12 and 45 ± 1.50 μM in AR positive (LNCaP) and negative (PC-3) cells, respectively. The reduced proliferation of PC-3 as well as LNCaP cells by conjugate correlated with accumulation of cells in G2/M phase and induction of caspase dependent apoptosis. Both PI3K/Akt and MAPK/ERK pathways played an important and differential role in conjugate-induced apoptosis of these PCa cells. While the inhibitor of Akt (A6730) or Akt-specific small interference RNA (siRNA) greatly sensitized PC-3 cells to conjugate-induced apoptosis, on the contrary, apoptosis was accelerated by inhibition of ERK (by PD98059 or ERK siRNA) in case of LNCaP cells, both ultimately culminating in the expression of cleaved caspase-3 protein. Moreover, anti-androgenic activity of the conjugate was mediated by decreased expression of AR and its co-activators (SRC-1, GRIP-1), thus interfering in their interactions with AR. All these data suggests that conjugate-induced inhibition of cell proliferation and induction of apoptosis are partly mediated by the down regulation of AR, Akt, and ERK signaling. These observations provide a rationale for devising novel therapeutic approaches for treating PCa by using conjugate alone or in combination with other therapeutics.

Show MeSH
Related in: MedlinePlus