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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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Induction of apoptosis by conjugate in prostate cancer cell line.(A) Structure of Resveratrol, Pterostilbene and Pterostilbene-isothiocyanate conjugate. (B) The effect of conjugate on the apoptosis of PC-3 and LNCaP cells as demonstrated by a representative FACS analysis using Annexin V as marker. (C) The histogram showing the data for FACS analysis where the results are the mean ± SEM of three independent experiments. # and *represents statistically significant difference with respect to their specific controls (vehicle treated) for cells in early and late apoptosis respectively at p<0.05.
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pone-0093335-g001: Induction of apoptosis by conjugate in prostate cancer cell line.(A) Structure of Resveratrol, Pterostilbene and Pterostilbene-isothiocyanate conjugate. (B) The effect of conjugate on the apoptosis of PC-3 and LNCaP cells as demonstrated by a representative FACS analysis using Annexin V as marker. (C) The histogram showing the data for FACS analysis where the results are the mean ± SEM of three independent experiments. # and *represents statistically significant difference with respect to their specific controls (vehicle treated) for cells in early and late apoptosis respectively at p<0.05.

Mentions: All cell culture reagents were obtained from GIBCO (Invitrogen, CA, USA), unless otherwise stated. Penicillin, streptomycin, MTT (3-(4, 5-dimethyl-2-thiazolyl) 2,5diphenyl-2H-tetrazoliumbromide), cell culture grade dimethyl sulphoxide (DMSO), agarose and all analytical grade chemicals were from HiMedia (Mumbai, India). Reverse transcription- polymerase chain reaction (RT-PCR) kits were from Genei (Bangalore, India). RESV, PTER, DHT, Akt1/2 kinase inhibitor, PD98059 (ERK inhibitor), Z-VAD-FMK (pan caspase inhibitor), Z-LEHD-FMK (caspase-9 specific inhibitor), Z-IETD-FMK (caspase-8 specific inhibitor) and BCA protein estimation kits were from Sigma-Aldrich (St. Louis, MO, USA). Polyfect transfection reagent was purchased from QIAGEN (Valencia, CA, USA). Pifithrin-α (p53 inhibitor), antibodies for caspase-3, Bax, Akt, p-Akt, ERK, p-ERK, SRC-1, GRIP-1, N-CoR, β-actin and small interfering RNAs (siRNAs) against Akt (sc-43609), ERK (sc-35335) and control (sc-37007; negative control for experiments using targeted siRNA transfection; each consists of a scrambled sequence that will not lead to the specific degradation of any known cellular mRNA) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PTER-ITC conjugate was synthesized in the asymmetric synthesis laboratory of the Department of Chemistry, Indian Institute of Technology Roorkee, India, according to the procedure described earlier [25] and henceforth designated as conjugate in the manuscript (Fig. 1A).


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)

Induction of apoptosis by conjugate in prostate cancer cell line.(A) Structure of Resveratrol, Pterostilbene and Pterostilbene-isothiocyanate conjugate. (B) The effect of conjugate on the apoptosis of PC-3 and LNCaP cells as demonstrated by a representative FACS analysis using Annexin V as marker. (C) The histogram showing the data for FACS analysis where the results are the mean ± SEM of three independent experiments. # and *represents statistically significant difference with respect to their specific controls (vehicle treated) for cells in early and late apoptosis respectively at p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0093335-g001: Induction of apoptosis by conjugate in prostate cancer cell line.(A) Structure of Resveratrol, Pterostilbene and Pterostilbene-isothiocyanate conjugate. (B) The effect of conjugate on the apoptosis of PC-3 and LNCaP cells as demonstrated by a representative FACS analysis using Annexin V as marker. (C) The histogram showing the data for FACS analysis where the results are the mean ± SEM of three independent experiments. # and *represents statistically significant difference with respect to their specific controls (vehicle treated) for cells in early and late apoptosis respectively at p<0.05.
Mentions: All cell culture reagents were obtained from GIBCO (Invitrogen, CA, USA), unless otherwise stated. Penicillin, streptomycin, MTT (3-(4, 5-dimethyl-2-thiazolyl) 2,5diphenyl-2H-tetrazoliumbromide), cell culture grade dimethyl sulphoxide (DMSO), agarose and all analytical grade chemicals were from HiMedia (Mumbai, India). Reverse transcription- polymerase chain reaction (RT-PCR) kits were from Genei (Bangalore, India). RESV, PTER, DHT, Akt1/2 kinase inhibitor, PD98059 (ERK inhibitor), Z-VAD-FMK (pan caspase inhibitor), Z-LEHD-FMK (caspase-9 specific inhibitor), Z-IETD-FMK (caspase-8 specific inhibitor) and BCA protein estimation kits were from Sigma-Aldrich (St. Louis, MO, USA). Polyfect transfection reagent was purchased from QIAGEN (Valencia, CA, USA). Pifithrin-α (p53 inhibitor), antibodies for caspase-3, Bax, Akt, p-Akt, ERK, p-ERK, SRC-1, GRIP-1, N-CoR, β-actin and small interfering RNAs (siRNAs) against Akt (sc-43609), ERK (sc-35335) and control (sc-37007; negative control for experiments using targeted siRNA transfection; each consists of a scrambled sequence that will not lead to the specific degradation of any known cellular mRNA) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PTER-ITC conjugate was synthesized in the asymmetric synthesis laboratory of the Department of Chemistry, Indian Institute of Technology Roorkee, India, according to the procedure described earlier [25] and henceforth designated as conjugate in the manuscript (Fig. 1A).

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