In the present study, we examined the chemopreventive effects and mechanisms of action of resveratrol on the androgen-responsive human prostate cancer cell LNCaP in cell culture and in a xenograft model. Our results suggest that resveratrol exerts differential effects in these in vitro and in vivo models.
Resveratrol exerted growth inhibitory effects in our cell culture model (). This is consistent with observations for resveratrol in similar in vitro
) and appeared to be due in part to modulation of androgen- and estrogen-responsive pathways, as we found that resveratrol inhibited both synthetic androgen R1881-induced and 17β-estradiol-induced LNCaP cell growth ( and ). The actions of resveratrol on these two pathways were further supported by our microarray and RT–PCR analyses of gene expression. Our microarray results indicated that resveratrol exerts a global effect on ARG mRNA expression ( and ), confirming findings by Jones et al.
). As in the cell growth experiment, resveratrol’s action on ARG mRNAs appeared to involve modulation of androgen- and estrogen-responsive pathways. Resveratrol inhibited both androgen and estrogen induction of ARG mRNA in LNCaP cells ( and ). The effect of resveratrol on ARG mRNAs correlated with expression at the protein level, as we showed resveratrol inhibited the R1881- and 17β-estradiol-induced increases in PSA protein levels (), similar to those reported by Hsieh et al.
). These molecular results are consistent with recent reports of in vitro
effects of resveratrol (41
) and provide further support for modulation of steroid hormone-dependent events as potential mechanisms that contribute to the overall growth inhibitory effects of resveratrol on LNCaP cells. Resveratrol is known to have weak estrogenic activity (16
), but in our experiments resveratrol appeared to act mainly as an antiestrogen, as resveratrol inhibited both 17β-estradiol-induced growth and increases in ARG mRNA levels. Interestingly, our binding study result (), as well as that reported by others (16
), suggests that resveratrol may not exerts its actions on androgen- and estrogen-mediated effects through direct competition of binding of steroid hormones to their receptors. Additional research is needed to elucidate the precise mechanisms of action.
Data presented here also show a novel effect of resveratrol on expression of steroid hormone-regulated genes in the LNCaP model. Resveratrol selectively reduced PSA, STK39 and IGF-1R ( and ) mRNAs in cells cultured in 10% FBS, but not expression of two other ARGs, B2M and SEPP1 (). We reason that the presence of estrogen-like activity in cell culture medium may explain these observations. Consistent with this interpretation, we found a lack of effects on expression of B2M and SEPP1 in the presence of externally added 17β-estradiol but not in the presence of the synthetic androgen R1881 in cells cultured in medium that is free of phenol red, a weak estrogen, and contains 10% CDS. Thus, culture conditions may greatly affect the interpretation of resveratrol’s biological activities.
In addition to the steroid hormone-responsive pathways, modulation of p53- and IGF-1-dependent events by resveratrol was also observed, as we found by microarray and confirmed by RT–PCR that resveratrol modulated both CDKN1A and IGF-1R mRNA levels. However, activation of the p53 pathway appeared to require a higher concentration (25 μM), whereas regulation of the steroid hormone and IGF-1 pathways appeared to have a lower threshold (1–5 μM). Thus, resveratrol may exert effects on multiple pathways in a concentration-dependent fashion. Given that, we measured circulating levels of resveratrol no higher than ~5 μM in the plasma of our animals, it is probably that modulation of steroid hormone–IGF-1-mediated events may be the more physiologically relevant mechanisms.
As illustrated in , we observed that exposure to resveratrol can lead to downregulation of IGF-1R and FRAP1–mTOR mRNAs but to upregulation of PIK3R3 mRNA. All three genes have been reported to be upstream regulators of AKT activity (36
). Activation of IGF-1R and FRAP/mTOR upregulates AKT activity (36
), whereas PIK3R3 downregulates AKT activity (37
). Modulation of IGF-1R, PIK3R3 and FRAP1–mTOR by resveratrol as reported here is consistent with recent reports of inhibitory effects of resveratrol on AKT-mediated pathways (41
). These results and our findings on modulation of androgen- and estrogen-mediated expression by resveratrol ( and ) lend further support for a recent report on modulation of androgen- and estrogen-mediated activation of AKT by resveratrol (41
). More importantly, we provide evidence that additional upstream molecular targets of AKT pathways such as IGF-1R, FRAP/mTOR and PIK3R3 are also modulated by resveratrol through androgen–estrogen-related events.
Our IHC results on expression of PSA, an ARG–estrogen-responsive gene, in xenograft tumors confirm the cell culture results and support our hypothesis that modulation of androgen- and estrogen-mediated pathways may contribute to resveratrol’s prostate cancer preventive effect in vivo
. Tumors from animals fed RES50 and RES100 diets had lower numbers of cells expressing PSA, corresponding to the observed delay in tumor growth in animals consuming these two diets. The effects on tumor growth and PSA protein expression appeared to be dose dependent ( and ). However, we also observed that not all cells within a tumor expressed similar levels of PSA. One possible explanation is that tumor cells in the xenograft may differ from the parent LNCaP cells, as LNCaP cells are known to undergo changes with different passages (44
). Interestingly, our animal experiment results are only partially correlated with the cell culture results. The lag in growth of tumor volume for animals consuming resveratrol appeared to ‘catch up’ to control animals by the seventh week (). Hence, modulation of the steroid hormone-dependent pathways may afford only partial protection against tumor development. Our IHC data on apoptosis and angiogenesis appear to suggest that resveratrol may not be entirely beneficial against tumor development. Tumors from animals fed resveratrol appeared to have lower apoptosis frequencies than animals on control diet. Moreover, tumors from the animals fed the higher dose of resveratrol (100 mg resveratrol/kg diet) also had significantly higher blood vessel counts, suggesting increased angiogenesis in the RES100 group compared with the control diet group. This effect of resveratrol on angiogenesis appeared not to be related to production of VEGF by tumor cells, as we did not observe qualitative differences in VEGF levels using IHC analysis.
The amount of resveratrol ingested by the animals per day (3–6 mg/day) in the present experiment is equivalent to consumption of 500–1000 ml of red wine per day and is readily achievable in humans (45
). By body surface area calculations (46
), our experimental animals consumed a dose of 12–24 mg/m2
of resveratrol and appeared to be at the lower end of pharmacological doses used in a recent human trial (47
). However, our dose was ~10× lower than that used in a recent study with TRAMP mice (48
). Interestingly, the plasma levels in our animals (1.3 ± 0.336 μM, RES100) were higher than those in Harper’s study [52 ± 18 nM, (48
)]. Hence, additional work would be necessary to extrapolate our results, as well as those from other animal models, to human populations.
In summary, we report here that resveratrol differentially affects in vivo and in vitro models of prostate cancer. In vitro, resveratrol appeared to exert growth inhibitory effects on cultured LNCaP cells through multiple pathways, including steroid hormone-dependent pathways. In vivo, resveratrol delayed the initial development of xenograft LNCaP cell tumors, consistent with an effect on steroid hormone-mediated events. However, exposure to resveratrol appeared to lead to promotion of angiogenesis and inhibition of apoptosis in LNCaP cell-derived tumors, as assessed by IHC markers.