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We described recently the growth inhibitory effects of the novel compound acyclic retinoid (ACR) in human hepatoma cell lines (M. Suzui et al., Cancer Res., 62: 3997–4006, 2002). In this study we examined the cellular and molecular effects of ACR on human squamous cell carcinoma (SCC) cells. ACR inhibited growth of the esophageal SCC cell line HCE7, and the head and neck SCC cell lines YCU-N861 and YCU-H891, with IC50 values of ∼10, 25, and 40 μm, respectively. Detailed studies were then done with HCE7 cells. Treatment of these cells with 10 μm ACR caused an increase of cells in G0-G1 and induced apoptosis. This was associated with two phases of molecular events. During phase 1, which occurred within 6–12 h, there was an increase in the retinoic acid receptor β (RARβ) and p21CIP1 proteins, and their corresponding mRNAs, and a decrease in the hyperphosphorylated form of the retinoblastoma protein. During phase 2, which occurred at ∼24 h, there was a decrease in the cellular level of transforming growth factor α, and the phosphorylated (i.e., activated) forms of the epidermal growth factor receptor, Stat3, and extracellular signal-regulated kinase proteins, and a decrease in both cyclin D1 protein and mRNA. Reporter assays indicated that ACR inhibited the transcriptional activity of the cyclin D1, c-fos, and activator protein promoters. On the other hand, ACR markedly stimulated the activity of a retinoic acid response element-CAT reporter when the cells were cotransfected with a RARβ expression vector. A hypothetical model explaining these two phases is presented. The diverse effects that we obtained with ACR suggest that this agent might be useful in the chemoprevention and/or therapy of human SCCs.

Overexpression of the HER-2/neu receptor (HER-2) is associated with a poor prognosis in patients with breast carcinoma and also in patients with head and neck squamous cell carcinoma (HNSCC). In a previous study on HNSCC cell lines, we found that epigalocathechin-3-gallate (EGCG), a major biologically active component of green tea, inhibited activation of the epidermal growth factor receptor (EGFR) and thereby inhibited EGFR-related downstream signaling pathways in HNSCC cells. In the present study, we examined the effects of EGCG on activation of the HER-2 receptor in human HNSCC and breast carcinoma cell lines that display constitutive activation of HER-2. Treatment of these cells with 10 or 30 microg of EGCG, respectively, doses that cause 50% inhibition of growth, markedly inhibited the phosphorylation of HER-2 in both cell lines. This was associated with inhibition of Stat3 activation, inhibition of c-fos and cyclin D1 promoter activity, and decreased cellular levels of the cyclin D1 and Bcl-XL proteins. Although these concentrations of EGCG are quite high, we found that concentrations of 0.1-1.0 microg/ml, which are in the range of plasma concentrations after administering a single oral dose of EGCG or a green tea extract, markedly enhanced the sensitivity of both types of cell lines to growth inhibition by Taxol, a drug frequently used in the treatment of breast carcinoma and HNSCC. These results, taken together with previous evidence that EGCG also inhibits activation of the EGFR in carcinoma cells, suggest that EGCG may be useful in treating cases of breast carcinoma and HNSCC in which activation of the EGFR and/or HER-2 plays important roles in tumor survival and growth.

We examined the effects of the phytochemical resveratrol in six human cancer cell lines (MCF7, SW480, HCE7, Seg-1, Bic-1, and HL60).Resveratrol induced marked growth inhibition in five of these cell lines, with IC(50) values of approximately 70-150 microM. However, only partial growth inhibition was seen in Bic-1 cells. After treatment with 300 microM resveratrol for 24 h, most of the cell lines were arrested in the S phase of the cell cycle. In addition, induction of apoptosis was demonstrated by the appearance of a sub-G(1) peak and confirmed using an annexin V-based assay. Cyclin B1 expression levels were decreased in all cell lines after 48 h of treatment. In SW480 cells, cyclin A, cyclin B1, and beta-catenin expression levels were decreased within 24 h. There was a decrease in cyclin D1 expression after only 2 h of treatment, and this persisted for up to 48 h. This decrease was partially blocked by concurrent treatment with the proteasome inhibitor calpain inhibitor I. Using a luciferase-based reporter assay, resveratrol did not inhibit cyclin D1 promoter activity in SW480 cells. Furthermore, using a reverse transcription-PCR-based assay, only a higher dose of resveratrol (300 microM) appeared to decrease cyclin D1 mRNA. Seg-1 cells expressed basal levels of cyclooxygenase-2 (cox-2), which was further induced by resveratrol. Neither basal levels nor induction of cox-2 was detectable in the remaining cell lines. Thus, cox-2 does not appear to be a critical target of this compound.These studies provide support for the use of resveratrol in chemoprevention and cancer therapy trials. Cyclin D1, cyclin B1, beta-catenin, and apoptotic index could be useful biomarkers to evaluate treatment efficacy.