Phytoestrogens are known to prevent tumor induction. But their molecular mechanisms of action are still unknown. This study aimed to examine the effect of apigenin on proliferation and apoptosis in HER2-expressing breast cancer cells. In our experiments, apigenin inhibited the proliferation of MCF-7 vec and MCF-7 HER2 cells. This growth inhibition was accompanied with an increase of sub G(0)/G(1) apoptotic fractions. Overexpression of HER2 did not confer resistance to apigenin in MCF-7 cells. Apigenin-induced extrinsic apoptosis pathway up-regulating the levels of cleaved caspase-8, and inducing the cleavage of poly (ADP-ribose) polymerase, whereas apigenin did not induce apoptosis via intrinsic mitochondrial apoptosis pathway since this compound did not decrease mitochondrial membrane potential maintaining red fluorescence and did not affect the levels of B-cell lymphoma 2 (BCL2) and Bcl-2-associated X protein. Moreover, apigenin reduced the tyrosine phosphorylation of HER2 (phospho-HER2 level) in MCF-7 HER2 cells, and up-regulated the levels of p53, phospho-p53 and p21 in MCF-7 vec and MCF-7 HER2 cells. This suggests that apigenin induces apoptosis through p53-dependent pathway. Apigenin also reduced the expression of phospho-JAK1 and phospho-STAT3 and decreased STAT3-dependent luciferase reporter gene activity in MCF-7 vec and MCF-7 HER2 cells. Apigenin decreased the phosphorylation level of IκBα in the cytosol, and abrogated the nuclear translocation of p65 within the nucleus suggesting that it blocks the activation of NFκB signaling pathway in MCF-7 vec and MCF-7 HER2 cells. Our study indicates that apigenin could be a potential useful compound to prevent or treat HER2-overexpressing breast cancer.

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Lung cancer is the leading cause of cancer deaths worldwide. Recent advances in targeted therapies hold promise for the development of new treatments for certain subsets of cancer patients by targeting specific signaling molecule. Based on the identification of the transcription factor cyclic AMP response element-binding protein (CREB) as an important regulator of growth of several types of cancers and our recent findings of its importance in normal differentiation of bronchial epithelial cells, we hypothesized that CREB plays an important pathobiologic role in lung carcinogenesis. We conducted this initial study to determine whether the expression and activation status of CREB are altered in non-small cell lung cancer (NSCLC) and of any prognostic importance in NSCLC patients. We found that the expression levels of mRNA and protein of CREB and phosphorylated CREB (p-CREB) were significantly higher in most of the NSCLC cell lines and tumor specimens than in the normal human tracheobronchial epithelial cells and adjacent normal lung tissue, respectively. Analysis of CREB mRNA expression and the CREB gene copy number showed that CREB overexpression occurred mainly at the transcriptional level. Immunohistochemical analysis of tissue microarray slides containing sections of NSCLC specimens obtained from 310 patients showed that a decreased survival duration was significantly associated with overexpression of CREB or p-CREB in never smokers but not in current or former smokers with NSCLC. These are the first reported results illustrating the potential of CREB as a molecular target for the prevention and treatment of NSCLC, especially in never smokers. [Cancer Res 2008;68(15):6065-73]

Phytoestrogens have been demonstrated to inhibit tumor induction; however, their molecular mechanisms of action have remained elusive. The present study aimed to investigate the effects of a phytoestrogen, apigenin, on proliferation and apoptosis of the human epidermal growth factor receptor 2 (HER2)-expressing breast cancer cell line SKBR3. Proliferation assay, MTT assay, fluorescence-activated cell sorting analysis, western blot analysis, immunocytochemistry, reverse transcription-polymerase chain reaction and ELISA assay were used in the present study. The results of the present study indicated that apigenin inhibited the proliferation of SKBR3 cells in a dose-and time-dependent manner. This inhibition of growth was accompanied by an increase in the sub-G0/G1 apoptotic population. Furthermore, apigenin enhanced the expression levels of cleaved caspase-8 and -3, and induced the cleavage of poly(adenosine diphosphate ribose) polymerase in SKBR3 cells, confirming that apigenin promotes apoptosis via a caspase-dependent pathway. Apigenin additionally reduced the expression of phosphorylated (p)-janus kinase 2 and p-signal transducer and activator of transcription 3 (STAT3), inhibited CoCl2-induced vascular endothelial growth factor (VEGF) secretion and decreased the nuclear localization of STAT3. The STAT3 inhibitor S31-201 decreased the cellular proliferation rate and reduced the expression of p-STAT3 and VEGF. Therefore, these results suggested that apigenin induced apoptosis via the inhibition of STAT3 signaling in SKBR3 cells. In conclusion, the results of the present study indicated that apigenin may be a potentially useful compound for the prevention or treatment of HER2-overexpressing breast cancer.

In a cell-free protein-synthesizing system from a rabbit reticulocyte lysate, total RNA extracted from cultured rat pituitary tumor (GH3) cells directed, in a dose-related manner, the synthesis of proteins that were precipitated by antisera specific to rat growth hormone (somatotropin) and rat prolactin. A marked decrease in growth hormone secretion and growth hormone mRNA activity was observed when cells were grown in a medium deficient in thyroid hormone. Addition of triiodothyronine in physiologic amounts both prevented and completely reversed this effect within 48 hr. Thyroid hormone had no effect on prolactin secretion or prolactin mRNA activity. These data suggest that thyroid hormone may stimulate synthesis of growth hormone throug induction of transcriptional activity. The possibility of an additional effect at the posttranscriptional level has not been excluded. Although thyroid hormone is believed to have a general effect on a variety of metabolic processes, some effects, at the molecular level, may be quite selective, as indicated by the observed changes in growth hormone but not prolactin mRNA activity. The GH3 cell model is useful in the study of triiodothyronine action because of independence from secondary hormonal effects caused by hypothyroidism and because simultaneous measurement of prolactin mRNA activity serves as a unique internal control. The demonstration of triiodothyronine (T3) binding to nuclear proteins raises the possibility that thyroid hormone may regulate gene expression. Earlier work from Tata's laboratory showed that thyroid hormone-induced protein synthesis was preceded by formation of new RNA (1). Later, DeGroot et al. and Dilman et al. showed increase in the poly(A)-rich fraction of RNA (2, 3). Demonstrations of stimulation of a specific mRNA by thyroid hormones were recently provided by Kurtz et al. (4) and by Roy et al. (5) for a2u-globulin in the rat. However, because a number of hormones are known to stimulate the synthesis of this protein (6) and because thyroid hormone profoundly alters the level of such hormones (7), experiments done in the whole animal do not provide sufficient evidence for the direct induction of -d2u-globulin mRNA by thyroid hormone. Samuels et al. have reported a quantitative correlation between nuclear T3 receptor occupancy and stimulation of growth hormone (GH, somatotropin) synthesis in cultured rat pituitary cells (8). We have adopted a similar experimental model to study more directly the action of thyroid hormone on the induction of a specific mRNA. In a recent report, we have shown that GH and prolactin mRNA activities in the total RNA extracted from a rat pituitary cell line (GH3) that actively synthesizes both hormones can be quantitated using a rabbit reticulocyte lysate cell-free system (9). In this report, we show that in the normal rat serum were 5.4 ,ug/100 ml and 60 ng/100 ml, respectively, and in the Tx rat serum were 0.6 ,g/100 ml and less than 20 ng/100 ml, respectively. RNA Extraction. Each RNA preparation was obta...

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BackgroundSilibinin is the major active molecule of silymarin, the mixture of flavonolignans extracted from Cirsium japonicum. It has been used for the treatment of hepatitis and inflammation-related diseases. In the present study, the effects of silibinin on allergic inflammation and its signaling were investigated in the induced human mast cells.MethodsCell growth inhibition induced by silibinin was measured by MTS assay. Histamine release was measured by enzyme immunoassay. The tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8) secreted protein levels and mRNA levels were measured by the ELISA assay and RT-PCR, respectively. The NF-κB promoter activity was examined by a luciferase assay.ResultsSilibinin suppressed the growth of HMC-1 cells and also reduced the production and mRNA expression of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-8. Moreover, silibinin inhibited the nuclear translocation of nuclear factor (NF)-κB through inhibition of the phosphorylation of IκBα and suppressed NF-κB transcriptional activity in stimulated HMC-1 cells.ConclusionsTaken together, these results indicate that silibinin inhibits the production of pro-inflammatory cytokines through inhibition of NF-κB signaling pathway in HMC-1 human mast cells, suggesting that silibinin could be used for the treatment of mast cell-derived allergic inflammatory diseases.

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