Abstract
Cocoa was shown to inhibit chemically induced carcinogenesis in animals and exert antioxidant activity in humans. However, the molecular mechanisms of the chemopreventive potential of cocoa and its active ingredient(s) remain unknown. Here we report that cocoa procyanidins inhibit neoplastic cell transformation by suppressing the kinase activity of mitogen-activated protein kinase kinase (MEK). A cocoa procyanidin fraction (CPF) and procyanidin B2 at 5 mug/ml and 40 mum, respectively, inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ mouse epidermal (JB6 P+) cells by 47 and 93%, respectively. The TPA-induced promoter activity and expression of cyclooxygenase-2, which is involved in tumor promotion and inflammation, were dose-dependently inhibited by CPF or procyanidin B2. The activation of activator protein-1 and nuclear factor-kappaB induced by TPA was also attenuated by CPF or procyanidin B2. The TPA-induced phosphorylation of MEK, extracellular signal-regulated kinase, and p90 ribosomal s6 kinase was suppressed by CPF or procyanidin B2. In vitro and ex vivo kinase assay data demonstrated that CPF or procyanidin B2 inhibited the kinase activity of MEK1 and directly bound with MEK1. CPF or procyanidin B2 suppressed JB6 P+ cell transformation induced by epidermal growth factor or H-Ras, both of which are known to be involved in MEK/ERK signal activation. In contrast, theobromine (up to 80 mum) had no effect on TPA-induced transformation, cyclooxygenase-2 expression, the transactivation of activator protein-1 or nuclear factor-kappaB, or MEK. Notably, procyanidin B2 exerted stronger inhibitory effects compared with PD098059 (a well known pharmacological inhibitor of MEK) on MEK1 activity and neoplastic cell transformation.
Highlights
Neoplastic transformation of cells is considered to be one of the major events occurring during carcinogenic processes
cocoa procyanidin fraction (CPF) or Procyanidin B2, but Not Theobromine, Inhibits MEK1 Kinase Activity—To investigate whether MEK1 might be a molecular target of CPF for the inhibition of cell transformation, we examined the effect of CPF on the kinase activity of MEK1
We investigated whether the molecular basis of the inhibition of cell transformation and COX-2 expression by procyanidin B2 is involved in the inhibition of MEK1 activity by CPF
Summary
Chemicals—Eagle’s minimum essential medium (MEM), basal medium Eagle (BME), gentamicin, and L-glutamine were purchased from Invitrogen; fetal bovine serum (FBS) was obtained from Gemini Bio-Products (Calabasas, CA); TPA and EGF were purchased from Sigma. CPF or Procyanidin B2, but Not Theobromine, Suppresses studies have shown that the MEK/ERK signaling pathway is TPA-induced COX-2 Promoter Activity and Protein Expression strongly involved in TPA-induced neoplastic transformation of in JB6 Pϩ Cells—A close relationship between chronic inflam- JB6 Pϩ cells [21, 28]. CPF or Procyanidin B2 Suppresses Either H-Ras- or EGF-induced Neoplastic Transformation of JB6 Pϩ Cells, Whereas Theobromine Has No Effect—Our previous studies showed that H-Ras and EGF activated the growth-signal pathway involving MEK and ERK protein kinases, leading to anchorage-independent growth of JB6 Pϩ cells [2, 22]. Based on the number of cell colonies, CPF at 40 g/ml inhibited H-Ras- or EGF-induced neoplastic cell transformation by 94 or 88%, respectively (Fig. 7, A and B) These findings provide evidence indicating that CPF suppresses cell transformation mainly by targeting the MEK/ERK signaling pathway. Together these findings provide evidence showing that procyanidin from cocoa suppresses cell transformation mainly by targeting the MEK/ERK signaling pathway
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