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In previous studies, we found that sulindac sulfide and exisulind (sulindac sulfone, Aptosyn) cause growth inhibition, arrest cells in the G1 phase of the cell cycle, and induce apoptosis in human breast cancer cell lines. These effects were associated with decreased expression of cyclin D1. The present study focuses on the effects of sulindac sulfide and exisulind on hormone signaling components in breast cancer cells. We found that estrogen receptor (ER)–positive and progesterone receptor (PR)–positive T47D breast cancer cells were somewhat more sensitive to growth inhibition by sulindac sulfide or exisulind than ER-negative PR-negative MB-MDA-468 breast cancer cells. Further studies indicated that sulindac sulfide and exisulind caused marked down-regulation of expression of the ER and PR-A and PR-B in T47D cells. However, neither compound caused a major change in expression of the retinoic acid receptor α (RARα), RARβ, or RARα in T47D cells. Sulindac sulfide and exisulind also caused a decrease in expression of the ER in estrogen-responsive MCF-7 breast cancer cells. Both compounds also markedly inhibited estrogen-stimulated activation of an estrogen-responsive promoter in transient transfection reporter assays. Treatment of T47D cells with specific protein kinase G (PKG) activators did not cause a decrease in ER or PR expression. Therefore, although sulindac sulfide and exisulind can cause activation of PKG, the inhibitory effects of these two compounds on ER and PR expression does not seem to be mediated by PKG. Our findings suggest that the growth inhibition by sulindac sulfide and exisulind in ER-positive and PR-positive human breast cancer cells may be mediated, in part, by inhibition of ER and PR signaling. Thus, these and related compounds may provide a novel approach to the prevention and treatment of human breast cancers, especially those that are ER positive.

Hepatoma is one of the most frequently occurring cancers worldwide. However, effective chemotherapeutic agents for this disease have not been developed. Acyclic retinoid, a novel synthetic retinoid, can reduce the incidence of postsurgical recurrence of hepatoma and improve the survival rate. OSI-461, a potent derivative of exisulind, can increase intracellular levels of cyclic GMP, which leads to activation of protein kinase G and induction of apoptosis in cancer cells. In the present study, we examined the combined effects of acyclic retinoid plus OSI-461 in the HepG2 human hepatoma cell line. We found that the combination of as little as 1.0 μmol/L acyclic retinoid and 0.01 μmol/L OSI-461 exerted synergistic inhibition of the growth of HepG2 cells. Combined treatment with low concentrations of these two agents also acted synergistically to induce apoptosis in HepG2 cells through induction of Bax and Apaf-1, reduction of Bcl-2 and Bcl-xL, and activation of caspase-3, -8, and -9. OSI-461 enhanced the G0-G1 arrest caused by acyclic retinoid, and the combination of these agents caused a synergistic decrease in the levels of expression of cyclin D1 protein and mRNA, inhibited cyclin D1 promoter activity, decreased the level of hyperphosphorylated forms of the Rb protein, induced increased cellular levels of the p21CIP1 protein and mRNA, and stimulated p21CIP1 promoter activity. Moreover, OSI-461 enhanced the ability of acyclic retinoid to induce increased cellular levels of retinoic acid receptor β and to stimulate retinoic acid response element-chloramphenicol acetyltransferase activity. A hypothetical model involving concerted effects on p21CIP1 and retinoic acid receptor β expression is proposed to explain these synergistic effects. Our results suggest that the combination of acyclic retinoid plus OSI-461 might be an effective regimen for the chemoprevention and chemotherapy of human hepatoma and possibly other malignancies.

Acyclic retinoid (ACR), a novel synthetic retinoid, has recently been demonstrated by us to inhibit the in vitro growth of human hepatoma cells, and this effect was associated with decreased expression of cell cycle-related molecules. These results, taken together with previous in vitro and clinical studies with ACR, suggest that this agent may be useful in the chemoprevention and therapy of hepatoma and possibly other human malignancies. In the present study, we further examined the molecular effects of ACR on the HepG2 human hepatoma cell line, focusing on the expression of nuclear retinoid receptors and the cell cycle inhibitor protein p21CIP1. Reverse transcription-PCR assays and Western blot analyses indicated that these cells express retinoic acid receptors (RARs) α, β, and γ, retinoid X receptors (RXRs) α and β, and peroxisome proliferator-activated receptors (PPAR) γ mRNA. Treatment with ACR caused a rapid induction within 3 h of RARβ mRNA and the related protein, but there was no significant change in the levels of the mRNA or proteins for RARs α and γ, RXRs α and β, and PPARγ. There was also a rapid increase in p21CIP1 mRNA and protein in HepG2 cells treated with ACR, and this induction occurred via a p53-independent mechanism. In transient transfection reporter assays, we cotransfected the retinoic acid response element-chloramphenicol acetyltransferase (CAT) reporter gene into HepG2 cells together with a RARβ expression vector. RARβ expression markedly stimulated CAT activity (up to about 4-fold) after the addition of ACR. However, CAT activity in the presence of ACR was only about 2-fold higher than that in the absence of ACR, when cells were cotransfected with RARs α and γ or RXRα. These findings suggest that the growth inhibitory effects of ACR are mediated at least in part through RARβ and that both RARβ and p21CIP1 play critical roles in the molecular mechanisms of growth inhibition induced by ACR.

The purpose of this study was to determine whether black cohosh contains constituents that inhibit the growth of human breast cancer cells, and therefore might eventually be useful in the prevention or treatment of breast cancer. Black cohosh rhizomes were extracted with methanol/water and fractionated by solvent-solvent partitioning to yield three fractions: hexane, ethyl acetate and water. The ethyl acetate fraction displayed the highest potency in two cell-based assays, growth inhibition and cell cycle analysis. This fraction inhibited growth of both the ER+ MCF7 and ER-MDA-MB-453 human breast cancer cell lines with IC50 values of about 20 and 10 micro g/ml, respectively. It also induced cell cycle arrest at G1 when tested at 30 micro g/ml and at G2/M at 60 micro g/ml in MCF7 cells. This suggests that the extract contains a mixture of components with the more active (or more abundant) causing G1 arrest and the less active causing G2/M arrest. We then examined specific components in this extract. The triterpene glycoside fraction obtained by polyamide column chromatography, and the specific triterpene glycosides actein, 23-epi-26-deoxyactein and cimiracemoside A, inhibited growth of the MCF7 human breast cancer cells and induced cell cycle arrest at G1. The most potent compound, actein, decreased the level of cyclin D1, cdk4 and the hyperphosphorylated form of the pRb protein and increased the level of p21cip1 in MCF7 cells, changes that may contribute to the arrest in G1. Further studies are in progress to identify the mechanisms by which actein and related compounds present in black cohosh inhibit growth of human breast cancer cells.

In a recent study on head and neck squamous cell carcinoma (HNSCC) cells we found that epigallocatechin-3-gallate (EGCG), a major biologically active component of green tea, inhibited activation of the epidermal growth factor receptor (EGFR) and related signaling pathways. Since activation of EGFR signaling pathways is associated with angiogenesis, we examined the effects of EGCG on vascular endothelial growth factor (VEGF) production by YCU-H891 HNSCC and MDA-MB-231 breast carcinoma cell lines, because we found that both of these cell lines display autocrine activation of transforming growth factor-alpha (TGF-alpha)/EGFR signaling and produce high levels of VEGF. Treatment with EGCG inhibited the constitutive activation of the EGFR, Stat3, and Akt in both cell lines. These changes were associated with inhibition of VEGF promoter activity and cellular production of VEGF. Mechanistic studies indicated that inhibition of Stat3, but not mitogen-activated protein kinase kinase (MEK)1 or phosphatidylinositol 3'-kinase (PI3K), significantly decreased VEGF promoter activity. However, the inhibitory effects of a dominant negative Stat3 on VEGF expression was not as strong as that produced by EGCG. An analysis of alternative pathways indicated that EGCG strongly inhibited the constitutive activation of NF-kappa B in both cell lines, and an NF-kappa B inhibitor strongly inhibited VEGF production. These results suggest that EGCG inhibits VEGF production by inhibiting both the constitutive activation of Stat3 and NF-kappa B, but not extracellular-signal-regulated kinase (ERK) or Akt, in these cells. Therefore, EGCG may be useful in treating HNSCC and breast carcinoma because it can exert both antiproliferative and antiangiogenic activities.

We examined the activity of two metabolites of sulindac (a nonsteroidal anti-inflammatory drug), sulindac sulfide and sulindac sulfone (exisulind, Prevatec), and a novel highly potent analog of exisulind (CP248) on a series of human prostate epithelial cell lines. Marked growth inhibition was seen with the BPH-1, LNCaP, and PC3 cell lines with IC50 values of about 66 microM, 137 microM, and 64 nM for sulindac sulfide, exisulind, and CP248, respectively. DNA flow cytometry and 4',6'-diamido-2-phenylindole (DAPI) staining indicated that these three compounds also induced apoptosis in all of these cell lines. Similar growth inhibition also was seen with the PrEC normal human prostate epithelial cell line, but these cells were resistant to induction of apoptosis at concentrations up to 300 microM, 1 mM, and 750 nM of sulindac sulfide, exisulind, and CP248, respectively. Derivatives of LNCaP cells that stably overexpress bcl-2 remained sensitive to growth inhibition and induction of apoptosis by these compounds. In vitro enzyme assays indicated that despite its high potency in inhibiting growth and inducing apoptosis, CP248, like exisulind, lacked cyclooxygenase (COX-1 and COX-2) inhibitory activity even at concentrations up to 10 mM. Moreover, despite variations of COX-1 and COX-2 expression, the three benign and malignant prostate cell lines showed similar sensitivity to growth inhibition and induction of apoptosis by these three compounds. Therefore, sulindac derivatives can cause growth inhibition and induce apoptosis in human prostate cancer cells by a COX-1 and -2 independent mechanism, and this occurs irrespective of androgen sensitivity or increased expression of bcl-2. These compounds may be useful in the prevention and treatment of human prostate cancer.

Purpose: (−)-Epigallocatechin gallate (EGCG) inhibits activation of the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor-2 (HER2) and multiple downstream signaling pathways in cancer cell lines. In this study we compared the cellular and molecular effects of EGCG with a well-standardized decaffeinated green tea catechin mixture Polyphenon E (Poly E) on human colon cancer cell lines. Experimental Design and Results: Both EGCG and Poly E preferentially inhibited growth of the Caco2, HCT116, HT29, SW480, and SW837 colon cancer cells when compared with the FHC normal human fetal colon cell line. The EGFR and HER2 proteins were overexpressed and constitutively activated in all of the colon cancer cell lines when compared with the FHC cell line. Treatment of HT29 cells with EGCG or Poly E caused an increase of cells in G1 and induced apoptosis. Both EGCG and Poly E caused a decrease in the phosphorylated forms of EGFR and HER2 proteins, and subsequently caused a decrease in the phosphorylated forms of the extracellular signal-regulated kinase and Akt proteins. Similar effects of these compounds were seen when the cells were stimulated with transforming growth factor α. Reporter assays indicated that both EGCG and Poly E inhibited the transcriptional activity of the activator protein 1 (AP-1), c-fos, nuclear factor κB, and cyclin D1 promoters. The combination of only 1 μg/mL of epicatechin plus 10 μg/mL of EGCG displayed synergistic effects on growth inhibition and induction of apoptosis. Furthermore, when treatment was prolonged for 96 hours, 1 μg/mL of EGCG or Poly E was sufficient to inhibit growth, reduce activation of EGFR and HER2, and induce apoptosis. Conclusion: Our findings suggest that EGCG or Poly E may be useful in the chemoprevention and/or treatment of colon cancer. Poly E contains about 60% EGCG, yet pure EGCG and Poly E had similar potencies (expressed as μg/ml). Poly E may be preferable because it is easier to prepare and this mixture of catechins may exert synergistic effects.

Acyclic retinoid (ACR), a novel synthetic retinoid, has recently been demonstrated by us to inhibit the in vitro growth of human hepatoma cells, and this effect was associated with decreased expression of cell cycle-related molecules. These results, taken together with previous in vitro and clinical studies with ACR, suggest that this agent may be useful in the chemoprevention and therapy of hepatoma and possibly other human malignancies. In the present study, we further examined the molecular effects of ACR on the HepG2 human hepatoma cell line, focusing on the expression of nuclear retinoid receptors and the cell cycle inhibitor protein p21(CIP1). Reverse transcription-PCR assays and Western blot analyses indicated that these cells express retinoic acid receptors (RARs) alpha, beta, and gamma, retinoid X receptors (RXRs) alpha and beta, and peroxisome proliferator-activated receptors (PPAR) gamma mRNA. Treatment with ACR caused a rapid induction within 3 h of RARbeta mRNA and the related protein, but there was no significant change in the levels of the mRNA or proteins for RARs alpha and gamma, RXRs alpha and beta, and PPARgamma. There was also a rapid increase in p21(CIP1) mRNA and protein in HepG2 cells treated with ACR, and this induction occurred via a p53-independent mechanism. In transient transfection reporter assays, we cotransfected the retinoic acid response element-chloramphenicol acetyltransferase (CAT) reporter gene into HepG2 cells together with a RARbeta expression vector. RARbeta expression markedly stimulated CAT activity (up to about 4-fold) after the addition of ACR. However, CAT activity in the presence of ACR was only about 2-fold higher than that in the absence of ACR, when cells were cotransfected with RARs alpha and gamma or RXRalpha. These findings suggest that the growth inhibitory effects of ACR are mediated at least in part through RARbeta and that both RARbeta and p21(CIP1) play critical roles in the molecular mechanisms of growth inhibition induced by ACR.

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.

In recent studies, we found that sulindac sulfide (SS), exisulind, CP248, and CP461 induce growth inhibition and apoptosis in a series of human prostate cancer cell lines, irrespective of cyclooxygenase expression, p53 mutations, or bcl-2 overexpression. Exisulind also inhibited the growth of the androgen-dependent LNCaP human prostate cancer cell line when grown as a xenograft in nude mice. This study demonstrates that doses of these compounds that induce growth inhibition and apoptosis in LNCaP cells also cause decreased prostate-specific antigen (PSA) secretion and decreased cellular levels of PSA. These effects appear to be a result, at least in part, of inhibition of the androgen receptor (AR) signaling pathway because the treated cells also display decreases in the level of the AR protein and mRNA and inhibition of transcription of an AR promoter luciferase reporter in transient transfection assays. SS and exisulind were more effective in inhibiting the expression of PSA and the AR than CP248 or CP461, apparently because of differential effects of these compounds on specific transcription factors. These findings suggest that the growth inhibition by these compounds in human prostate cancer cells may be mediated, in part, by inhibition of AR signaling. Thus, these compounds may provide a novel approach to the prevention and treatment of human prostate cancer.

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.

In a recent study on head and neck squamous cell carcinoma (HNSCC) cells we found that epigallocatechin-3-gallate (EGCG), a major biologically active component of green tea, inhibited activation of the epidermal growth factor receptor (EGFR) and related signaling pathways. Since activation of EGFR signaling pathways is associated with angiogenesis, we examined the effects of EGCG on vascular endothelial growth factor (VEGF) production by YCU-H891 HNSCC and MDA-MB-231 breast carcinoma cell lines, because we found that both of these cell lines display autocrine activation of transforming growth factor-alpha (TGF-alpha)/EGFR signaling and produce high levels of VEGF. Treatment with EGCG inhibited the constitutive activation of the EGFR, Stat3, and Akt in both cell lines. These changes were associated with inhibition of VEGF promoter activity and cellular production of VEGF. Mechanistic studies indicated that inhibition of Stat3, but not mitogen-activated protein kinase kinase (MEK)1 or phosphatidylinositol 3'-kinase (PI3K), significantly decreased VEGF promoter activity. However, the inhibitory effects of a dominant negative Stat3 on VEGF expression was not as strong as that produced by EGCG. An analysis of alternative pathways indicated that EGCG strongly inhibited the constitutive activation of NF-kappa B in both cell lines, and an NF-kappa B inhibitor strongly inhibited VEGF production. These results suggest that EGCG inhibits VEGF production by inhibiting both the constitutive activation of Stat3 and NF-kappa B, but not extracellular-signal-regulated kinase (ERK) or Akt, in these cells. Therefore, EGCG may be useful in treating HNSCC and breast carcinoma because it can exert both antiproliferative and antiangiogenic activities.

Acyclic retinoid (ACR), a novel synthetic retinoid, can prevent the recurrence of human hepatoma after surgical resection of primary tumors, but the molecular mechanisms by which ACR exerts antitumor effects are not known. In this study, we found that ACR inhibited the growth of three human hepatoma cell lines. In HepG2 cells, this inhibition was associated with an arrest of the cell cycle in G(0)-G(1), increased cellular levels of p21(CIP1), decreased levels of the hyperphosphorylated form of the retinoblastoma protein, and decreased levels of cyclin D1, but no significant changes were seen in the levels of the p16(INK4a), p27(KIP1), cyclin-dependent kinase 4, cyclin-dependent kinase 6, glycogen synthase kinase 3beta, or beta-catenin proteins. ACR also caused a decrease in the level of cyclin D1 mRNA. Cotreatment of HepG2 human hepatoma cells with the proteasome inhibitor N-acetyl-Leu-Leu-norleu-al did not prevent the ACR-induced decrease in cyclin D1 protein, in contrast to the protective effect of N-acetyl-Leu-Leu-norleu-al on the cyclin D1 protein in cells treated with all-trans-retinoic acid. In transient transfection reporter assays, ACR, but not all-trans-retinoic acid, inhibited transcription from the cyclin D1 promoter. As reported previously in colon carcinoma cells, we found that in hepatoma cells, cyclin D1 promoter activity is markedly stimulated by the beta-catenin/T-cell factor pathway. Nevertheless, even in the presence of excess beta-catenin, ACR markedly inhibited the transcriptional activity of the cyclin D1 promoter. This is the first systematic study of the inhibitory effects of ACR, or any other retinoid compound, on beta-catenin/T-cell factor-stimulated cyclin D1 promoter activity in human tumor cells. These novel effects of ACR provide further evidence that ACR may be a valuable agent in the chemoprevention and therapy of hepatoma and possibly other human malignancies.

Nonsteriodal anti-inflammatory drugs (NSAIDs) are among the most commonly used medications in the United States and elsewhere, mainly for the treatment of arthritis. The NSAID sulindac causes regression and prevents the recurrence of premalignant colonic polyps in patients with familial adenomatous polyposis and inhibits colon carcinogenesis in rodents. Sulindac and sulindac sulfone, a metabolite of sulindac that lacks cyclooxygenase (cox) inhibitory activity, also inhibit mammary carcinogenesis in rats. To obtain insights into the relevance of these findings to human breast cancer, we examined the mechanism of action of sulindac and its sulfide and sulfone metabolites on the normal human mammary epithelial cell line MCF-10F and the human breast cancer cell line MCF-7. Of the three compounds, the sulfide was the most potent inhibitor of cell growth, although the sulfone and sulfoxide were also active at higher concentrations. Treatment of MCF-10F and MCF-7 cells with 100 microM sulindac sulfide resulted in accumulation of cells in the G1 phase of the cell cycle and induction of apoptosis. Apoptosis occurred within 24 h as determined by the TUNEL assay and DNA laddering was observed at 72 h. The accumulation of cells in G1 was associated with decreased levels of expression of cyclin D1 but no effect was seen on the expression of CDK4 or the immediate early response gene c-jun. Treatment with sulindac sulfide caused a striking induction of the CDK inhibitor p21WAF1 in MCF-10F cells. The MCF-7 cell line expressed a high basal level of p21WAF1 which did not change significantly after drug treatment. The pro-apoptotic gene BAX was not induced in either MCF-10F or MCF-7 cells by sulindac sulfide. Stable overexpression of cyclin D1, which frequently occurs in breast cancers, did not protect mammary epithelial cells from inhibition by the sulfide. These studies suggest that this class of compounds warrants further study with respect to breast cancer prevention and treatment.