To investigate the ability of trans-R, cis-R, and dihydro-R to control the growth of PC-3 prostate cancer cells, various concentrations of these compounds were added to tumor cell cultures and then incubated for 48 h. Figures and show the tumor cell viability after exposure to these agents.
Biological activity of dihydro-R, trans-R and cis-R in the PC-3 cell line.
Biological activity of dihydro-R and trans-R in the DU-145 cell line.
The cytotoxic effect of trans-R was observed at concentrations of 1 × 10-7 to 1 × 10-4 M. Similarly, the cytotoxic effect of cis-R was observed at concentrations 1 × 10-6 to 1 × 10-4 M. The results indicated that trans-R is somewhat more effective as an anticancer agent than cis-R. Dihydro-R, in contrast to trans-R and cis-R, had a biphasic effect. At high concentrations (1 × 10-5 to 1 × 10-3 M), dihydro-R showed the typical for this class of compounds decrease in the growth rate of tumor cells. However, treatment with 1×10-10 to 1 × 10-7 M of dihydro-R significantly increased the growth of PC-3 prostate cancer cells (Figure ).
Further comparative evaluation of the biological activity of trans-R and dihydro-R was performed using the DU-145 prostate cancer cell line. Again, trans-R displayed only cytotoxic properties whereas dihydro-R had a biphasic effect, similar to the one observed in PC-3 cells. At high concentrations (1 × 10-5 to 1 × 10-3 M), dihydro-R decreased the rate of growth of DU-145 tumor cells. Treatment with low concentrations (1 × 10-10 to 1 × 10-8 M) of dihydro-R significantly increased the growth of DU-145 prostate cancer cells (Figure ).
The observed cytotoxic effects of both trans
in androgen-independent prostate cancer cell lines are in agreement with the previously reported data. Trans
is a known inhibitor of human prostate cancer cell growth at concentrations of 1 × 10-6
to 1 × 10-4
M (e.g., GI(50) = 2.41 × 10-5
M, DU-145 [8
]) depending on the prostate cancer cell line and experimental conditions [9
]. The biphasic action of the third form of R
, in comparison with the better studied trans
requires further evaluation, especially in relation to estrogen receptors [6
] present in both the PC-3 and the DU-145 prostate cancer cell lines [10
The biological effects of trans
in androgen-independent prostate cancer cell lines can be attributed to a variety of specific molecular targets, including estrogen and androgen receptors [15
]. Unfortunately, the exact mechanism of the hormone receptor-mediated activity of trans
is poorly understood because of the apparent complexity of the underlying biological functions of these receptors in prostate cancer cells. For example, it is well documented that 17β-estradiol (E2) inhibits growth of androgen-independent PC-3 cells [10
] but stimulates growth of androgen-dependent LNCaP cells, presumably via its own receptor [16
]. The situation is even further complicated by the existence of two estrogen receptors (ERs), ER-α and ER-β, in prostate cancer cells [11
]. As a result, growth of PC-3 cells, which have both ER subtypes, can be effectively inhibited by estrogens as well as antiestrogens [17
]. In contrast, DU-145 cells express only ER-β, and their growth can be inhibited only by antiestrogens [11
Given the convolution of hormone receptor functions in prostate cancer cells [18
], it could only be presented as a working hypothesis that, similar to MCF-7 breast cancer cells [6
], the observed growth stimulation of PC-3 and DU-145 cells by nanomolar concentrations of dihydro
can be attributed to the hormone receptor modulation activity of this compound. A direct comparison between the three cell lines (MCF-7 [6
], PC-3, and DU-145, Figure ) reveals substantial similarities in dihydro
biphasic proliferative action. As expected, the proliferative effects are more pronounced in the "classic" ER-α-dependent MCF-7 cells compared to PC-3 cells (ER-α and ER-β) and then DU-145 cells (only ER-β).
Biological activity of dihydro-R in PC-3, DU-145, and MCF-7 cell lines.
Additional credence is given to our assumption that dihydro
is a hormone receptor modulator by the recent observation that another dietary polyphenol with a similar biological profile, genistein (Figure ), also exhibits biphasic behavior in the regulation of prostate cancer cell growth [19
]. It was reported that low (5 × 10-7
M) concentrations of genistein caused increased proliferation of PC-3 cells whereas a pharmacologic dose (5 × 10-5
M) decreased proliferation. Estrogen signaling is mentioned as one of the possible mechanisms of this biphasic action [19
] (genistein is a well-known phytoestrogen [14
To explore our working hypothesis further, we evaluated the cancer cell growth effects of dihydro
in the presence of trans
is capable of acting as both an estrogen and an antiestrogen [22
], including non-genomic action [25
], but this compound by itself does not significantly affect the growth of PC-3 prostate cancer cells at concentrations below 1 × 10-7
M. Our experiments clearly demonstrated the interference between dihydro
(Figure ). In these experiments trans
acted as a dihydro
antagonist at low concentrations. The maximum antagonistic effect was observed at nanomolar concentrations (1 × 10-10
to 1 × 10-8
M). Similar biphasic agonistic/antagonistic properties of trans
in MCF-7 cells in the presence of E2 were reported previously [24
Biological activity of dihydro-R and trans-R mixtures (1/1; 10/1; 1/10) in PC-3 cells.
Somewhat stronger interference between dihydro-R
was observed in "classic" ER-α-dependent MCF-7 cells (Figure ). As we mentioned previously, dihydro-R
proliferative action at very low concentrations was more pronounced in the MCF-7 cell line than the PC-3 cell line (Figure ). As a result, cross-modulation effects of dihydro-R
in MCF-7 cells were notable even at sub-picomolar concentrations (1 × 10-16
to 1 × 10-13
M). This modulation appeared to be non-monotonic and showed a very complex concentration dependence pattern. For example, maximum proliferation effects of a 1/1 mixture of dihydro-R
were observed at 1 × 10-14
and 1 × 10-7
M concentrations, whereas intermediate concentrations (1 × 10-11
to 1 × 10-9
M) showed little, if any, proliferative activity (Figure ). Non-monotonic dose response is a well-documented feature of steroid hormone receptors [27
Biological activity of trans-R and dihydro-R mixtures (1/1; 1/10; 10/1) in MCF-7 cells line.