Suppression of Src/ERK and GSK-3/ß-catenin signaling by pinosylvin inhibits the growth of human colorectal cancer cells.

Pinosylvin, a naturally occurring trans-stilbenoid mainly found in Pinus species, has exhibited a potential cancer chemopreventive activity. However, the growth inhibitory activity against cancer cells and the underlying molecular mechanisms remain to be elucidated. Therefore, the anti-proliferative activity of pinosylvin was investigated in human colorectal HCT 116 cancer cells. Pinosylvin inhibited the proliferation of HCT 116 cells by arresting transition of cell cycle from G1 to S phase along with the downregulation of cyclin D1, cyclin E, cyclin A, cyclin dependent kinase 2 (CDK2), CDK4, c-Myc, and retinoblastoma protein (pRb), and the upregulation of p21(WAF1/CIP1) and p53. Pinosylvin was also found to attenuate the activation of proteins involved in focal adhesion kinase (FAK)/c-Src/extracellular signal-regulated kinase (ERK) signaling, and phosphoinositide 3-kinase (PI3K)/Akt/ glycogen synthase kinase 3ß (GSK-3ß) signaling pathway. Subsequently, pinosylvin suppressed the nuclear translocation of ß-catenin, one of downstream molecules of PI3K/Akt/GSK-3ß signaling, and these events led to the sequential downregulation of ß-catenin-mediated transcription of target genes including BMP4, ID2, survivin, cyclin D1, MMP7, and c-Myc. These findings demonstrate that the anti-proliferative activity of pinosylvin might be associated with the cell cycle arrest and downregulation of cell proliferation regulating signaling pathways in human colorectal cancer cells.

Antimetastatic activity of pinosylvin, a natural stilbenoid, is associated with the suppression of matrix metalloproteinases.

Metastasis is a major cause of death in cancer patients. Our previous studies showed that pinosylvin, a naturally occurring trans-stilbenoid mainly found in Pinus species, exhibited a potential cancer chemopreventive activity and also inhibited the growth of various human cancer cell lines via the regulation of cell cycle progression. In this study, we further evaluated the potential antimetastatic activity of pinosylvin in in vitro and in vivo models. Pinosylvin suppressed the expression of matrix metalloproteinase (MMP)-2, MMP-9 and membrane type 1-MMP in cultured human fibrosarcoma HT1080 cells. We also found that pinosylvin inhibited the migration of HT1080 cells in colony dispersion and wound healing assay systems. In in vivo spontaneous pulmonary metastasis model employing intravenously injected CT26 mouse colon cancer cells in Balb/c mice, pinosylvin (10 mg/kg body weight, intraperitoneal administration) significantly inhibited the formation of tumor nodules and tumor weight in lung tissues. The analysis of tumor in lung tissues indicated that the antimetastatic effect of pinosylvin coincided with the down-regulation of MMP-9 and cyclooxygenase-2 expression, and phosphorylation of ERK1/2 and Akt. These data suggest that pinosylvin might be an effective inhibitor of tumor cell metastasis via modulation of MMPs.

Nuclear factor E2-related factor 2-mediated induction of NAD(P)H:quinone oxidoreductase 1 by 3,5-dimethoxy-trans-stilbene.

NAD(P)H:quinone oxidoreductase 1 (NQO1), a phase II enzyme, plays an important role in the detoxification or chemoprotection of carcinogens, and induction of this enzyme is a target for the prevention of carcinogenesis. Natural stilbenoids have potential cancer chemopreventive activities, potentially through affecting NQO1 activity. Along this line, several stilbenoids were evaluated to procure more potent compounds for inducing NQO1 activity in cultured murine Hepa 1c1c7 cells. As a result, we found that 3,5-dimethoxy-trans-stilbene (DMS) possesses potent NQO1 induction activity through up-regulation of both protein and mRNA expression of NQO1 as determined by Western blot and reverse transcription-polymerase chain reaction analysis, respectively. DMS also increased protein expression of heme oxygenase-1 (HO-1), another phase II enzyme. This induction of NQO1 and HO-1 by DMS was closely related to the regulation of nuclear factor E2-related factor 2 (Nrf2). The translocation and activation of Nrf2 by DMS was also involved in the modulation of the upstream signal transduction molecule, protein kinase C δ. These findings suggest that DMS might have a cancer chemopreventive activity by inducing detoxifying enzymes such as NQO1 and HO-1.

Pinosylvin suppresses LPS-stimulated inducible nitric oxide synthase expression via the MyD88-independent, but TRIF-dependent downregulation of IRF-3 signaling pathway in mouse macrophage cells.

Since inhibitors of inducible nitric oxide synthase (iNOS) have been considered as potential anti-inflammatory and cancer chemopreventive agents, we have evaluated the inhibitory effects on the production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells with natural and synthetic compounds. Pinosylvin (3,5-dihydroxy-trans-stilbene), a stilbenoid mainly found in heartwood of Pinus sylvestris, exhibited the inhibition of iNOS protein and mRNA expression. The plausible mechanisms of pinosylvin on the suppression of iNOS gene expression were found to be associated with the downregulation of interferon regulatory factor 3 (IRF-3) and interferon-ß (IFN-ß) expression, which are related to Toll/IL-1 receptor domain-containing adapter inducing interferon-ß (TRIF)-mediated signaling. Decreased IFN-ß expression suppressed a phosphorylation of JAK kinase, and subsequently, the phosphorylation of signal transducer and activator of transcription-1, one of the iNOS transcriptional activators, was inhibited by pinosylvin. In addition, the suppression of poly(I:C)-induced iNOS expression, and the attenuation of iNOS expression under the IRF-3 gene knock-down condition also confirmed that pinosylvin affects TRIF pathway. These findings demonstrate that the suppression of LPS-induced iNOS expression by pinosylvin is associated with the regulation of MyD88-independent, but TRIF-mediated signaling pathway.

Synthesis and inhibitory effects of pinosylvin derivatives on prostaglandin E2 production in lipopolysaccharide-induced mouse macrophage cells.

A series of natural stilbenoids, pinosylvin and its derivatives, were synthesized and evaluated for the inhibitory activity of prostaglandin E(2) production in lipopolysaccharide-induced RAW 264.7 cells. Potential inhibitors, including 3,5-dimethoxy-trans-stilbene and 3-hydroxy-5-benzyloxy-trans-stilbene, have been newly identified, and thus providing chemical leads for the further development of anti-inflammatory or cancer chemopreventive agents.