Our group has previously reported the dual effects of resveratrol in ER(-) breast cancer cell migration and invasion, while others have shown that resveratrol functions as an anti-estrogen in ER(+) mammary cancer cell lines and rodent models
]. Our studies with ERα(-), ERβ(+) MDA-MB-231 breast cancer cell line demonstrated that resveratrol at 50 μM acts in an anti-estrogenic manner to reduce cell migration while, resveratrol at 5 μM acts similar to estrogen inducing cell migration, invasion, and formation of lamellipodia
]. Lamellipodia are actin structures found at the leading edge of migrating cells that are under Rac regulation
]. We reported that 50 μM resveratrol decreases Rac activity, whereas estrogen and 5 μM resveratrol increases Rac activity in breast cancer cells. Moreover, using breast cancer cells expressing dominant-negative Rac, we demonstrated that signaling to the actin cytoskeleton by low concentrations of resveratrol was under Rac regulation
]. Since, breast cancers often lose ER expression during progression, we tested the effects of resveratrol in both ERβ(+) and ER(-) metastatic breast cancer cells MDA-MB-231 and MDA-MB-435 in vivo
Many in vivo studies supporting the anti-cancer effects of resveratrol have used high, non-physiologically rele-vant, concentrations of resveratrol of 50-800 mg/kg BW
]. However, the effects of low concentrations of resveratrol, which are critical for a better understanding of the possible effect of dietary resveratrol in cancer and for dosing recommendations, have been poorly addressed at the in vivo scenario. Therefore low, physiologically relevant, concentrations of resveratrol (0.5 and 5 mg/kg BW) and a moderate concentration (50 mg/kg BW) were used to test resveratrol’s effect on the progression of breast cancer.
The bioavailability of resveratrol is considered to be low as per data on plasma levels of resveratrol following dietary consumption. Studies have shown that administration of 20 mg/kg resveratrol to rats, mice, or rabbits demonstrated a very short half-life (10 minutes) and reached circulating levels of less than 1 μM very rapidly
]. Mice administered 100 mg/kg of resveratrol achieved a maximum serum concentration of 2277 ng/mL at 0.25 h and ~42 ng/mL at 24 h
]. However, others have shown that, administration of as little as 50 μg/kg body weight of resveratrol per day for 3 months to rats achieved serum levels as high as 8.0 μM
]. In the present study, we administered low to moderate (0.5, 5, and 50 mg/kg BW) concentrations of resveratrol daily 5× a week for 1.5 and 4 months in the MDA-MB-435 and MDA-MB231 studies, respectively. Therefore, we expect the resveratrol levels in the plasma to be low but consistent, and higher in the mammary tissue, as per previous studies that have shown resveratrol levels in plasma are low due to high absorption by tissues
In SCID mice with MDA-MB-231 mammary fat pad tumors, oral gavage of resveratrol resulted in a nonsignificant, but evident increase in tumor growth of ~2-fold at 0.5 mg/kg BW resveratrol and ~2.5-fold at 5 and 50 mg/kg BW resveratrol (Figure
A, B, and C). Similarly, the growth of mammary fat pad tumors established in nude mice from the more aggressive and highly metastatic breast cancer cell line MDA-MB-435 was increased by resveratrol at all the concentrations tested. In the MDA-MB-435 study, the concentration that showed the greater effect was 0.5 mg/kg BW resveratrol with a ~3-fold increase, whereas 5 and 50 mg/kg BW resveratrol increased tumor growth by ~2.5-fold (Figure
D, E, and F). This increase in tumor growth presented with a p
>0.05 compared to controls.
This high probability may be due to the heterogeneity of individual tumor growth, because when we re-group into growing and poorly growing groups as cited in
there was a significant difference of 0.02 in the 5 mg/kg BW resveratrol group compared to vehicle controls. Moreover, administration of resveratrol at all concentrations tested did not result in significant differences in mouse weight among the resveratrol treated groups compared to vehicle controls (Additional file
: Figure S1).
Figure 1 Effect of resveratrol on the growth of mammary fat pad tumors. 1 × 106 GFP-tagged MDA-MB-231 or MDA-MB-435 cells in Matrigel:DMEM (1:1) were inoculated at the mammary fat pad of female SCID (MDA-MB-231) or athymic nude mice (MDA-MB-435). One week (more ...)
Even though our previous study, where we administered 5 mg/kg BW resveratrol in combination with equal amounts of quercetin and catechin, demonstrated reduced tumor growth and metastasis
], in this study we found resveratrol alone induced a marked trend of increase in tumor growth, especially by the lower concentrations; thus, indicating a deleterious effect of resveratrol on breast cancer progression. This may suggest an alternate mechanism where resveratrol alone acts to promote tumor growth.
Our results highlight the importance of attaining a better understanding of the effects of resveratrol at low concentrations. Also noteworthy is the fact that 50 mg/kg BW resveratrol, which in other studies has been shown to reduce tumor growth in ovarian cancer and hepatocarcinoma
], induced tumor growth in both (MDA-MB-231 and MDA-MB-435) breast cancer cells. This suggests that resveratrol effects are not only concentration dependent but can also vary depending on the cell and cancer type.
To investigate the role of resveratrol on breast cancer metastasis, lungs, livers, kidneys, and bones from mice bearing MDA-MB-231 and MDA-MB-435 mammary tumors were harvested at necropsy for identification of fluorescent metastatic foci. Mice from the MDA-MB-231 study only presented with lung metastasis; whereas, mice from the MDA-MB-435 study presented with metastasis to all harvested organs due to the highly metastatic potential of this cell line. Resveratrol dramatically induced lung metastasis in both studies. In mice bearing MDA-MB-231 derived mammary tumors, resveratrol at 0.5, mg/kg BW caused a 30-fold increase, and at 5 and 50 mg/kg BW, a significant 13- and 65-fold increase in lung metastasis after 108 days (Figure
). Likewise, in mice bearing MDA-MB-435 tumors, resveratrol at 0.5, 5, and 50 mg/kg BW caused an 11-, 36-, and 9-fold increase in lung metastasis, respectively, after only 44 days (Figure
). In the case of kidneys and bone, mice from the vehicle group did not present with metastasis while resveratrol-treated mice developed metastases (Table
). Metastasis to the kidneys was observed in 14%, 10%, and 22% of mice in 0.5, 5, and 50 mg/kg BW resveratrol treatment groups, respectively; whereas, metastasis to the bone was observed in 20% and 22% of mice treated with 5 and 50 mg/kg BW resveratrol, respectively. Finally, liver metastases were identified in a similar percentage of mice from each treatment group, including vehicle control. However, the average integrated density was 10- and 2.9- fold higher in 0.5 and 5 mg/kg BW resveratrol treated mice (Table
), suggesting a higher number and/or larger metastatic foci in mice treated with low concentrations of resveratrol when compared to vehicle treated mice. Overall, the percentage of mice presented with metastasis was low for statistical comparisons due to the short duration of the study which, because of high tumor burden, had to be terminated after only 44 days. Regardless, our findings strongly suggest that resveratrol at low concentrations can promote mammary tumor growth and metastasis.
Figure 2 Effect of resveratrol on lung metastasis. Following necropsy, lungs were excised from mice with GFP-MDA-MB-231 or GFP-MDA-MB-435 mammary tumors that received vehicle or 0.5, 5, or 50 mg/kg BW Res diets and analyzed for metastases by fluorescent microscopy (more ...)
Effects of resveratrol on metastasis of GFP-MDA-MB-435 mammary tumors
Since resveratrol-mediated regulation of breast cancer cell migration/invasion was previously shown by us to be dependent on signaling from the small GTPase Rac
], we investigated Rac activation status in MDA-MB-231 and MDA-MB-435-derived mammary tumors. In MDA-MB-231 mammary tumors, resveratrol at 50 mg/kg BW significantly increased Rac activity by 3.5-fold; whereas 0.5 and 5 mg/kg BW caused a non-significant increase in Rac activity of 1.75 and 2-fold, respectively (Figure
). On the other hand, in MDA-MB-435-derived tumors, resveratrol at 0.5 and 50 mg/kg BW significantly increased Rac activity by 1.6- and 1.8-fold, respectively (Figure
). Our results suggest that resveratrol-mediated induction of metastasis might be, at least partly due to increased Rac activity.
Figure 3 Rac activity in resveratrol treated tumors. GFP-MDA-MB-231 or GFP-MDA-MB-435 mammary tumors from mice that received vehicle or 0.5, 5, or 50 mg/kg BW Res diets were lysed. PAK–PBD–GST beads were used to pull down active (GTP-bound) Rac (more ...)
We also investigated the effects of resveratrol in the tumoral expression of Akt and the MAPK jun kinase (JNK), key proteins in survival signaling and cell proliferation that are commonly disregulated in human cancers and have been shown to be regulated by resveratrol [4,6;10-12;38-40]. In addition, we studied the expression of the Rac downstream effector p-21 activated kinase 1 (PAK1), implicated in the regulation of cell motility/migration and therefore, metastasis. PAKs are also relevant for the control of other cellular processes important for tumorigenesis such as survival, proliferation and gene transcription
], via regulation of Akt and MAPKs pathways
]. We analyzed Akt, JNK, and PAK1 protein expression in MDA-MB-231 derived mammary tumors by western blot. Our results show a trend in upregulation (1.75-2.5-fold) of these kinases in tumors from mice that received resveratrol at 5 and 50 mg/kg BW; thus, implicating the PI3-K/Akt, MAPK, and Rac/PAK pathways in potential effects of resveratrol on mammary tumor growth and metastasis (Figure
Figure 4 Effect of resveratrol on Akt, JNK, and PAK expression in mammary tumors. Total lysates of MDA-MB-231-derived mammary tumors from vehicle- and Res-treated mice were western blotted with anti-Akt, anti-JNK, or anti-PAK1 antibodies. Fold changes in protein (more ...)
To test whether the increased expression of Akt, JNK, and PAK1 resulted in parallel increases in their activity, we tested the effect of low concentrations of resveratrol on the active phosphorylated levels of these signaling proteins. Quiescent MDA-MB-231 or MDA-MB-435 cells were treated with 0.1, 0.5, and 5 μM resveratrol for 15 min and the lysates western blotted using total or phsopho-specific antibodies to Akt, JNK, and PAK1. In parallel with increased expression of total proteins from the MDA-MB-231 mammary tumors in mice treated with resveratrol (Figure
), we observed increased phosphorylation of Akt, JNK, and PAK1 by resveratrol at all concentrations tested in MDA-MB-231 cells (Figure
), but not in MDA-MB-435 cells (data not shown). Therefore, the upregulated expression and activities of the Rac effectors PAK, JNK, and Akt, are expected to contribute to the observed increase in tumor growth and metastasis in response to resveratrol.
Figure 5 Effect of resveratrol on Akt, JNK, and PAK1 activity in MDA-MB-231 breast cancer cells. Confluent MDA-MB-231 cells were serum starved for 24 h, treated with Veh, 0.1, 0.5, or 5 μM resveratrol (Res) for 15 min, lysed immediately, and western blotted (more ...)
Even though resveratrol at high concentrations inhibits Akt and MAPK activities, at low concentrations, resveratrol has been previously shown to promote proliferation in human cancer cells and to induce Akt and MAPKs, among other tumorigenic signaling proteins
]. Resveratrol modulates a plethora of signaling pathways important for tumor progression; and therefore, it would be too simplistic to limit resveratrol’s cancer promoting (or inhibiting) properties to a few molecules or a single signaling pathway
]. Thus, it is more reasonable to think that the collective activity of resveratrol, rather than a single effect, is responsible for our results. In addition to modulation of steroid receptor signaling and crosstalk with growth factor receptors that can affect both signaling and transcriptional activities
], resveratrol was recently shown to exhibit dose dependent regulation of Wnt signaling and the expression of Wnt target genes
]. Since resveratrol affected mammary tumor growth in both ERα(-), ERβ(+) MDA-MB-231 and ER(-) MDA-MB-435 cancer cells, ER independent mechanisms may be responsible for our intriguing findings. Such mechanisms can include: 1) the regulation of ER related receptors that are active in ER(-) breast cancers
] and are regulated by phytoestrogens
], or 2) the collective regulation of multiple signaling and transcriptional pathways.