A major challenge in current cancer research is to identify novel approaches to chemoprevention using non-toxic natural substances. RES is a natural phytoalexin found in various dietary constituents including the skin of red grapes. We previously showed that RES can inhibit cell cycle progression A431 cells (G1/S phase) lacking functional p53 (Kim et al., 2006
). In this study, we show that oral administration of RES to p53+/−
/SKH-1 transgenic mice results in p53-independent suppression of UVB-induced skin carcinogenesis. Specifically, UVB-induced SCCs appeared later in the RES-treated mice, and the malignant conversion of benign papillomas to SCCs was substantially inhibited. RES treatment resulted in increased expression of E-cadherin and decreased TGF-β2 expression in SCCs.
In probing the mechanism(s) underlying TGF-β2 downregulation, we found that RES inhibited both TGF-β2/Smad-dependent and -independent ERK signaling pathways and suppressed the in vitro
invasiveness of A431 cells. RES inhibited TGF-β2 expression, which is known to be regulated by CREB transcriptional factors. The CREB/ATF family of transcription factors responds to various signals that drive cell proliferation, differentiation, and adaptive responses (Shaywitz and Greenberg, 1999
). In addition to cAMP-dependent protein kinase A (PKA), CREB is a target of various kinases, including Akt1 and mitogen- and stress-activated kinase 1, a kinase activated in the p38 MAPK- and ERK-dependent cascades (Caravatta et al., 2008
; Deak et al., 1998
; Xing et al., 1996
). RES-mediated Akt1 inhibition occurred concomitant with decreased CREB phosphorylation. Furthermore, by constitutively activating Akt it was possible to partially rescue RES inhibition of CREB phosphorylation and TGF-β2 expression, indicating that RES suppression of TGF-β2 involves downregulation of the Akt/CREB pathway.
TGF-β is important in modulating tumor progression (Cui et al., 1996
; Han et al., 2005
; Valcourt et al., 2005
). At least three TGF-β isoforms have been identified (TGF-β1, TGF-β2, and TGF-β3). Among these, TGF-β2 overexpression has been observed in more advanced stages of gastric carcinoma and is thought to correlate with a poor prognosis (Vagenas et al., 2007
). It has been reported that specific inhibition of TGF-β2 using an antisense oligodeoxynucleotide prolongs the survival time of patients suffering from recurrent or refractory malignant gliomas, including two patients in whom long-lasting complete tumor remission occurred (Hau et al., 2007
). These observations suggest that identifying effective methods for TGF-β2 inhibition/downregulation may be therapeutically efficacious in later stages of tumor progression. In skin, it is possible that each TGF-β isoform could have differential effects during various stages of carcinogenesis. TGF-β1 expression appears to be associated with a more differentiated tumor phenotype, whereas TGF-β2 expression is more typical of highly malignant and invasive phenotypes. TGF-β3 expression is enhanced in the stroma of malignant tumors in subcutaneously growing HaCat (immortalized human keratinocytes)-ras
tumors in nude mice (Gold et al., 2000
). Our results are consistent with this pattern in that TGF-β2 was less strongly overexpressed in UVB-induced benign precursors (papillomas) of SCCs than in UVB-induced SCCs, implying that TGF-β2 may drive the latter stages of UVB-induced skin carcinogenesis. RES reduced TGF-β2 expression both in vitro
and in vivo
. It should be pointed out that in contrast to our data; RES was previously shown to increase the transcription of the TGF-β2 gene and the TGF-β2 protein expression, and to activate Smad signaling in an autocrine manner in the A549 human lung epithelial cell line (Suenaga et al., 2008
). However, lower doses (3–10 μM) of RES were used this study, and the effect of these dosages on TGF-β2 induction was not correlated to cell viability or other cellular functions. In another study RES (10μM) inhibited TGF-β2 in breast cancer cells (Serrero and Lu, 2001
). These differential effects may be due to differences in the RES concentrations used or to differential effects in various tissue types. In our in vitro
study, RES downregulated both TGF-β/Smad and Smad-independent ERK activation. Our data indicate that RES-mediated inhibition of ERK activation is likely mediated through TGF-β2, since exogenous addition of TGF-β2 but not TGF-β1 rescued ERK activation. Interestingly, Smad-2 inhibition was not rescued by the addition of TGF-β2 or -β1, suggesting TGF-β-independent mechanisms for the RES-mediated suppression of Smad2 activation. Although TGF-β signaling is known to promote EMT via both Smad and non-Smad dependent pathways (Zavadil and Bottinger, 2005
), mice with a keratinocyte-specific Smad2 deletion reportedly have accelerated formation and malignant progression of chemically induced skin tumors, and Smad2-deficient tumors exhibit EMT (Hoot et al., 2008
). In our studies in A431 cells, RES had no effect on Smad2. It is not clear in the present study whether Smad2 deficiency and inhibition of Smad2 phosphorylation have different functions.
EMT blockade offers a potentially novel and effective approach for the prevention/treatment of cancer, particularly in lesions with a propensity for metastasis. However, accelerated EMT may be only a partial determinant of malignant progression that may require other oncogenic events to promote tumor invasion. Other pathways commonly activated in late-stage skin carcinogenesis, such as Akt and NF-κB, are known to enhance EMT (Dong et al., 2007
; Hoot et al., 2008
; Lester et al., 2007
). Here we have shown that RES inhibits Akt-mediated TGF-β2 functional cooperation between cellular growth signaling and TGF-β pathway regulators, which may enhance EMT. Whether there is a spatial correlation (i.e., expression in the invasion front) between TGF-β2/Akt expression and tumor progression, or whether and how RES specifically targets these cells in vivo
requires further investigation. In summary, our data indicate that RES is a potent inhibitor of tumor cell growth and may modulate tumor progression by altering the EMT process by downregulating TGF-β2.