TGFbeta dependent polyamine depletion is associated with cell growth inhibitory effects of isothiocyanate sulforaphane in colon cancer cells

Introduction: Sulforaphane (SFN), a naturally occurring isothiocyanate present in cruciferous vegetables, is an attractive agent due to its potent anticancer effects. SFN was shown to suppress the proliferation of various cancer cells in vitro and in vivo, whereas the underlying molecular mechanisms remain largely unknown. Previous studies could demonstrate that modulation of polyamine metabolism provides a chemopreventive strategy of different phytochemicals. Thus, the objective of this study was to elucidate a possible regulation of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, by SFN in a cell culture model of colorectal cancers.

Methods: Caco-2 and SW620 cells were cultures under standard conditions. Cell growth was determined by BrdU incorporation and crystal violet staining. Protein levels of TGFβ and phospho-Smad were examined by Western blot analysis. TGFβ secretion was measured in cell culture supernatants by a commercially available ELISA. Ornithine decarboxylase activity was assayed radiometrically measuring [¹⁴CO₂] liberation.

Results: Sulforaphane [5–50µM] inhibits cell growth significantly both in Caco-2- and SW620 cells in a dose and time dependent manner (***p<0.001). Antiproliferative effects of Sulforphane closely correlate with a dose-dependent reduction of ODC activity (˜50% at [50µM], ***p<0.001) after 24h. This was further confirmed by the addition of exogenous polyamine spermine [5µM] and spermidine [10µM], which significantly counteracted cell growth inhibitory effects of SNF after 24h. Furthermore, sulforaphane causes a concentration dependent induction of TGFβ protein expression after 2 to 6h (˜100% at [50µM]) and TGFβ secretion after 24h as well as a prominent phosphorylation of Smad2 (˜40% at [50µM], a downstream component of TGFβ signaling. Moreover, co-treatment with TGFβ receptor kinase inhibitor SB431542 [10µM] largely abolished inhibitory effects of SFN on ODC activity.

Conclusion: These data provide evidence for the involvement of TGFβ signaling in SFN mediated inhibition of ODC activity and thus polyamine depletion in colorectal cancer cells.