Cytotoxicity induced by resveratrol, pterostilbene and conjugate in noncancer (COS-1, CHO) and prostate cancer (PC-3, LNCaP) cell lines after 24 h exposure as determined by MTT assay.

a<p>50% growth inhibition as determined by MTT assay (24 h drug exposure).</p>b<p>Compound tested in triplicate, data expressed as mean value ± SEM of three independent experiments.</p>c<p><i>p</i><0.001 between resveratrol and conjugate treated group.</p>d<p><i>p</i><0.001 between PTER and conjugate treated group.</p

Pterostilbene-Isothiocyanate Conjugate Suppresses Growth of Prostate Cancer Cells Irrespective of Androgen Receptor Status

<div><p>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.</p></div

Transcriptional and translational analysis of various apoptotic marker genes in response to conjugate.

<p>Expression patterns of various apoptotic marker genes in response to varying doses of conjugate treatment as determined by RT-PCR in (A) PC-3 and (B) LNCaP cells. (C) Immunoblot analysis of various apoptotic marker genes in response to different doses of conjugate and resveratrol in PC-3 and (D) LNCaP cells. The histogram on the right panel of each figure represents densitometric analyses of the image data and expressed as percent of control in conjugate treated cells respectively where the results are mean ± SEM of three independent experiments. *represents statistically significant difference with respect to control for each genes at <i>p</i><0.05.</p

Role of p53 protein on conjugate induced cell death in LNCaP cells.

<p>Effect of p53 inhibitor (PFT-α) on (A) the expression of apoptotic genes as determined by immunoblot analysis and (B) cell death in conjugate treated LNCaP cells as determined by MTT assay. Data are the mean ± SEM of three independent experiments. # and *indicates significant difference with respect to the controls for p53 and caspsase-3 proteins respectively at <i>p</i><0.05. (C) Immunoblot analysis to show the phosphorylation patterns of Akt and ERK in PC-3 and (D) LNCaP cells in response to varying doses of conjugate and resveratrol treatments. The histogram on the right panel of each figure represents densitometric analyses of the image data and expressed as percent of control where the results are mean ± SEM of three independent experiments. *represents statistically significant difference with respect to control for each group at <i>p</i><0.05.</p

Conjugate inhibits androgen receptor transactivation and translocation in prostate cancer cells.

<p>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 (<i>p</i><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.</p

The conjugate induces G2/M phase arrest and caspase dependent cell death in prostate cancer cells.

<p>Cell cycle distribution of (A) PC-3 and (B) LNCaP cells upon treatment with varying doses of conjugate. Results are presented as mean ± SEM of three independent experiments. *represents statistically significant difference with respect to vehicle treated PC-3 and LNCaP cells respectively corresponding to each stage of cell cycle at <i>p</i><0.05. (C) The effects of varying doses of conjugate (left panel) and resveratrol (right panel) on caspase-8, -9 and -3 activities in PC-3 and (D) LNCaP cells. Results are the mean ± SEM of three independent experiments. *represents statistically significant difference with respect to control cells for respective caspases tested at <i>p</i><0.05. (E) The effect of caspase inhibitors on conjugate-induced cell death in PC-3 and (F) LNCaP cells. The cells were pre-treated with 20 μM of respective caspase inhibitors: Z-LEHDFMK (caspase-9 inhibitor); Z-IETDFMK (caspase-8 inhibitor); and Z-VAD-FMK (general caspase inhibitor) for 1 h before the addition of 20 μM conjugate. Cell death was measured 24 h after conjugate treatment using MTT assay. Data are the mean ± SEM of three independent experiments. *and # represents statistically significant difference with respect to control (vehicle) and conjugate treated cells respectively for each cell lines at <i>p</i><0.05. ns, non-significant.</p

Effect of conjugate on androgen receptor expression in LNCaP cells.

<p>(A) Regulation of androgen receptor expression in LNCaP cells by varying doses of conjugate as determined by RT-PCR. (B) Immunoblot analysis to show the expression of androgen receptor in response to conjugate and (C) resveratrol. The histogram on the right panel of each figure represents densitometric analyses of the image data and expressed as percent of control where the results are mean ± SEM of three independent experiments. *and # represents statistically significant difference with respect to control and 10 nM DHT respectively at <i>p</i><0.05. (D) Immunofluorescence analysis to show the expression of androgen receptor (400× magnification). (E) Effect of conjugate on androgen receptor protein turnover in LNCaP cells. LNCaP cells were grown to 60–70% confluency and treated with 10 μg/ml protein synthesis inhibitor cycloheximide (30 min pre-treatment) with or without 10 μM conjugate at 0 h. Cells were then harvested at various time points (0, 3, 6 12, 24 and 36 h) and lysates were prepared. Androgen receptor protein levels were determined by immunoblot analysis using anti-androgen receptor antibody and normalized to β-actin control. The results are representative of two independent experiments. CHX, cycloheximide.</p

Conjugate weakens the interaction between androgen receptor and its co-activators.

<p>(A) Immunoblot analysis for the expression of androgen receptor co-regulators in response to conjugate in LNCaP cells and the histogram on the right panel of the figure represents densitometric analyses of the image data and expressed as percent of control in conjugate treated cells where the results are mean ± SEM of three independent experiments. *represents statistically significant difference with respect to control (<i>p</i><0.05). (B) Effects of conjugate on the interactions of androgen receptor and its co-activators (SRC-1 and GRIP-1) as determined by co-immunoprecipitation analysis. Histogram in the lower panel represents ratio of androgen receptor to that of co-activators where results are mean ± SEM of three independent experiments. *represents statistically significant difference with respect to control (<i>p</i><0.05). (C) Effect of conjugate on DHT induced transactivation of androgen receptor in presence/absence of co-regulators (SRC-1 and GRIP-1) in androgen receptor positive LNCaP cells. Luciferase activities are expressed as percentage of transactivation with respect to only DHT treated group which is considered as 100%. *and # indicates statistically significant difference from DHT treated cells in absence and presence of co-regulators respectively and **indicates significant level of difference between two adjacent groups with and without co-activator at <i>p</i><0.05. ns, non-significant.</p

Proposed scheme for conjugate mediated actions on LNCaP and PC-3 prostate cancer cells.

<p>Conjugate inhibits both androgen receptor and ERK signalling and finally contributing to its downstream effects of decreased cell viability and increased apoptosis in androgen receptor positive LNCaP cells. In case of androgen receptor negative PC-3 cells, inhibition of Akt and its downstream targets contributes to conjugate mediated decrease in cell viability and increased apoptosis.</p

Differential role of PI3K/Akt and MAPK/ERK pathways in conjugate induced apoptosis of prostate cancer cells.

<p>Effects of siRNA mediated silencing of Akt and ERK on conjugate-induced apoptosis of (A) PC-3 and (B) LNCaP cells. LNCaP and PC-3 PCa cells were transfected with siRNAs (at a final concentration of 100 nM) using polyfect transfection reagent. After 24 h of transfection the cells were treated with 20 μM conjugate and allowed to grow for another 24 h. The cell lysates were prepared and the level of p-Akt, p-ERK and cleaved caspase-3 proteins were detected by immunoblot analysis. The histogram on the right panel of each figure represents densitometric analyses of the image data and expressed as percent of control where the results are mean ± SEM of three independent experiments. *and # represents statistically significant difference with respect to control and 20 μM conjugate treated groups respectively at <i>p</i><0.05.</p