Daih-Huang Kuo

Resveratrol Modulates Tumor Cell Proliferation and Protein Translation via SIRT1-Dependent AMPK Activation

Resveratrol functions as an agonist for estrogen receptor (ER)-mediated transcription. However, other researchers have reported that resveratrol decreases proliferation of breast cancer cells that are either ER-positive or ER-negative, which suggests that the interaction of resveratrol with the ER may not fully explain its inhibitory effect on proliferation. Similar to those effects associated with caloric restriction (CR), resveratrol has multiple beneficial activities, such as increased life span and delay in the onset of diseases associated with aging. One key enzyme thought to be activated during CR is the AMP-activated kinase (AMPK), a sensor of cellular energy levels. The suppression of nonessential energy expenditure by activated AMPK along with the CR mimetic and antiproliferative properties of resveratrol has led us to hypothesize that resveratrol activity might have an important role in the activation of AMPK. Here, we show that resveratrol activated AMPK in both ER-positive and ER-negative breast cancer cells. Once activated, AMPK inhibited 4E-BP1 signaling and mRNA translation via mammalian target of rapamycin (mTOR). Moreover, we also found that AMPK activity mediated by resveratrol in cancer cells was due to inducing the expression of Sirtuin type 1 (SIRT1) via elevation in the cellular NAD(+)/NADH in ER-positive cells. To our knowledge, we demonstrate here for the first time that resveratrol induces the expression of SIRT1 protein in human cancer cells. These observations raise the possibility that SIRT1 functions as a novel upstream regulator for AMPK signaling and may additionally modulate tumor cell proliferation. Targeting SIRT1/AMPK signaling by resveratrol may have potential therapeutic implications for cancer and age-related diseases.

Inhibitory effect of magnolol on TPA-induced skin inflammation and tumor promotion in mice.

Magnolol has been reported to have an anti-inflammatory and antitumor effect in vitro and in vivo. Herein, we report the investigation of the inhibitory effects of magnolol on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in mouse skin. We found that the topical application of magnolol effectively inhibited the transcriptional activation of iNOS and COX-2 mRNA and proteins in mouse skin stimulated by TPA. Pretreatment with magnolol resulted in the reduction of TPA-induced nuclear translocation of the nuclear factor-kappaB (NFkappaB) subunit and DNA binding by blocking the phosphorylation of IkappaBalpha and p65 and subsequent degradation of IkappaBalpha. In addition, magnolol can suppress TPA-induced activation of extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)/Akt, which are upstream of NFkappaB. Moreover, magnolol significantly inhibited 7,12-dimethylbene[a]anthracene (DMBA)/TPA-induced skin tumor formation by reducing the tumor multiplicity, tumor incidence, and tumor size of papillomas at 20 weeks. All these results revealed that magnolol is an effective antitumor agent and that its inhibitory effect is through the down-regulation of inflammatory iNOS and COX-2 gene expression in mouse skin, suggesting that magnolol is a novel functional agent capable of preventing inflammation-associated tumorigenesis.

Inhibition of epidermal growth factor receptor signaling by Saussurea involucrata, a rare traditional Chinese medicinal herb, in human hormone-resistant prostate cancer PC-3 cells.

Prostate carcinoma is the most frequently diagnosed malignancy and the second leading cause of death of men in the United States. To date, no effective therapeutic treatment allows abrogation of the progression of prostate cancer to more invasive forms. In this study, we identified Saussurea involucrata Kar. et Kir., a rare traditional Chinese medicinal herb, as a potential agent for androgen-independent prostate cancer patients and investigated its biological mechanism as an antineoplastic agent. S. involucrata caused a concentration- and time-dependent inhibition of cell proliferation in human hormone-resistant prostate cancer PC-3 cells. Moreover, in vitro studies in a panel of several types of human cancer cell lines revealed that S. involucrata inhibited cell proliferation with high potency. To evaluate the bioactive compounds, we successively extracted the S. involucrata with fractions of methanol (SI-1), ethyl acetate (SI-2), n-butanol (SI-3), and water (SI-4). Among these extracts, SI-2 contains the most effective bioactivity. SI-2 treatment resulted in significant time-dependent growth inhibition together with G1 phase cell cycle arrest and apoptosis in PC3 cells. In addition, SI-2 treatment strongly induced p21WAF1/CIP and p27KIP1 expression, independent of the p53 pathway, and downregulated expression of cyclin D1 and cyclin-dependent kinase 4 (CDK4). SI-2 treatment increased levels of Bax, cytochrome c, activated caspase-3, and active caspase-9 and decreased Bcl-2 expression level. One of the major targets for the therapy in prostate cancer can be epidermal growth factor receptor (EGFR). SI-2 markedly reduced phosphorylation of EGFR and inhibited activation of AKT and STAT3. Moreover, p.o. administration of SI-2 induced a dose-dependent inhibition of PC-3 tumor growth in vivo. In summary, our study identifies S. involucrata as an effective inhibitor of EGFR signaling in human hormone-resistant prostate cancer PC-3 cells. We suggest that S. involucrata could be developed as an agent for the management of EGFR-positive human cancers.

Osthole Suppresses Hepatocyte Growth Factor (HGF)-Induced Epithelial-Mesenchymal Transition via Repression of the c-Met/Akt/mTOR Pathway in Human Breast Cancer Cells

Substantial activation of the HGF/c-Met signaling pathway is involved in the progression of several types of cancers and associated with increased tumor invasion and metastatic potential. Underlying HGF-induced tumorigenesis, epithelial to mesenchymal transition (EMT) shows a positive correlation with progression in patients. We previously determined that osthole is a potent fatty acid synthase (FASN) inhibitor. FASN is implicated in cancer progression and may regulate lipid raft function. We therefore examined whether osthole could block HGF-induced tumorigenesis by disrupting lipid rafts. Here, we found that osthole could abrogate HGF-induced cell scattering, migration, and invasion in MCF-7 breast cancer cells. Osthole also effectively inhibited the HGF-induced decrease of E-cadherin and increase of vimentin via down-regulation of phosphorylated Akt and mTOR. Interestingly, osthole blocked HGF-induced c-Met phosphorylation and repressed the expression of total c-Met protein in MCF-7 cells. In addition, C75, a pharmacological inhibitor of FASN, repressed the expression of total c-Met protein in MCF-7 cells. Consistent with a role for FASN, loss of c-Met in cells treated with osthole was prevented by the exogenous addition of palmitate. Briefly, our result suggests a connection between FASN activity and c-Met protein expression and that osthole is a potential compound for breast cancer therapy by targeting the major pathway of HGF/c-Met-induced EMT.

Indoleamine 2,3-Dioxygenase, an Immunomodulatory Protein, Is Suppressed by (−)-Epigallocatechin-3-gallate via Blocking of γ-Interferon-Induced JAK-PKC-δ-STAT1 Signaling in Human Oral Cancer Cells

Immune escape is a characteristic of cancer progression, but its underlying molecular mechanism is still poorly understood. An immunomodulatory protein, indoleamide 2,3-dioxygenase (IDO), is induced by gamma-interferon (IFN-gamma) in several immune cells; those cells are observed in cancer cell microenvironment and can enhance immune escape. Previous studies show that IDO is expressed in the process of tumor formation and associated with cancer cell immune tolerance. By locally degrading tryptophan, IDO inhibits the proliferation of T lymphocytes and induces T cell apoptosis, leading to suppression of T cell response. In this study, (-)-epigallocatechin-3-gallate (EGCG), the major constituent of green tea, is found to significantly inhibit the expression of IDO in human oral cancer cell lines. EGCG suppresses the induction of IDO at transcriptional level. Activation of STAT1 is discovered to play an important role in regulating IDO expression by IFN-gamma. The study results demonstrate that EGCG can inhibit translocation of STAT1 into nucleus in IFN-gamma-stimulated human oral cancer cells. In addition, both tyrosine and serine phosphorylation of STAT1 are revealed to be suppressed by EGCG. Moreover, phosphorylation of PKC-delta, JAK-1, and JAK-2, which are the upstream event for the activation of STAT1, are also inhibited by EGCG in IFN-gamma-stimulated human oral cancer cells. These data show that EGCG inhibited IDO expression by blocking the IFN-gamma-induced JAK-PKC-delta-STAT1 signaling pathway. This study indicates that EGCG is a potential drug for immune and target therapy to enhance cancer therapy by increasing antitumor immunity.

Effect of ShanZha, a Chinese herbal product, on obesity and dyslipidemia in hamsters receiving high-fat diet

AIM The present study is designed to investigate the effect of shanzha (Crataegus pinnatifida) on obesity or dyslipidemia induced by high-fat diet in hamsters and characterize the role of PPARalpha in this action of shanzha. MATERIALS AND METHODS We induced dyslipidemia and obesity in hamsters using high-fat diet and treated hamsters with shanzha or vehicle for 7 days. We measured the body weight, adipose tissue weights, and food intake of hamsters. Plasma total cholesterol (TC), triglyceride (TG), LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C) were determined at the beginning and end of this treatment. Effect of shanzha on adipogenesis was examined in vitro and change of PPARalpha was analyzed using Western blot. RESULTS The food intake, body weights, and weights of both brown and white adipose tissues were markedly reduced in hamsters receiving shanzha as compared with the vehicle-treated control. Plasma levels of TC, TG and LDL-C were decreased by this shanzha treatment while HDL-C was elevated. The effects of shanzha were reversed by the combined treatment with PPARalpha antagonist, MK886. Shanzha inhibited the fat droplet accumulation in 3T3-L1 adipocytes in a dose-dependent manner and this effect was abolished by MK886. Western blot results showed activation of PPARalpha by shanzha in hamster adipose tissue. CONCLUSION We suggest that shanzha could activate PPARalpha to improve dyslipidemia or obesity.

Curcumin-loaded nanoparticles enhance apoptotic cell death of U2OS human osteosarcoma cells through the Akt-Bad signaling pathway

Curcumin has potential anticancer activity and has been shown to be involved in several signaling pathways including differentiation and apoptosis. Our previous study showed that water-soluble PLGA curcumin nanoparticles (Cur-NPs) triggered apoptotic cell death through regulation of the function of MDR1 and the production of reactive oxygen species (ROS) in cisplatin-resistant human oral cancer CAR cells. In this study, we investigated the anti-proliferative effects of Cur-NPs on human osteosarcoma U2OS cells. The morphology of Cur-NPs showed spherical shape by TEM analysis. The encapsulation efficiency of curcumin in Cur-NPs prepared by single emulsion was 90.5 ± 3.0%. Our results demonstrated that the curcumin fragments on the mass spectrum of Cur-NPs and the peaks of curcumin standard could be found on the Cur-NPs spectrum by 1H-NMR spectra analysis. Cur-NPs induced anti-proliferative effects and apoptosis in U2OS cells. Compared to the untreated U2OS cells, more detectable amount of Cur-NPs was found inside the treated U2OS cells. Cur-NPs induced DNA fragmentation and apoptotic bodies in U2OS cells. Both the activity and the expression levels of caspases-3/-7 and caspase-9 were elevated in the treated U2OS cells. Cur-NPs upregulated the protein expression levels of cleaved caspase-3/caspase-9, cytochrome c, Apaf-1 and Bad and downregulated the protein expression level of p-Akt in U2OS cells. These results suggest Cur-NPs are effective in enhancing apoptosis in human osteosarcoma cells and thus could provide potential for cancer therapeutics.

Induction of Apoptosis by [8]-shogaol via Reactive Oxygen Species Generation, Glutathione Depletion and Caspase Activation in Human Leukemia Cells

Ginger, the rhizome of Zingiber officinale , is a traditional medicine with a carminative effect and antinausea, anti-inflammatory, and anticarcinogenic properties. This study examined the growth inhibitory effects of [8]-shogaol, one of the pungent phenolic compounds in ginger, on human leukemia HL-60 cells. It demonstrated that [8]-shogaol was able to induce apoptosis in a time- and concentration-dependent manner. Treatment with [8]-shogaol caused a rapid loss of mitochondrial transmembrane potential, stimulation of reactive oxygen species (ROS) production, release of mitochondrial cytochrome c into cytosol, and subsequent induction of procaspase-9 and procaspase-3 processing. Taken together, these results suggest for the first time that ROS production and depletion of glutathione that contributed to [8]-shogaol-induced apoptosis in HL-60 cells.

Allyl isothiocyanate inhibits cell metastasis through suppression of the MAPK pathways in epidermal growth factor‑stimulated HT29 human colorectal adenocarcinoma cells.

Allyl isothiocyanate (AITC) has been found to present sources from consumed cruciferous vegetables. AITC is known to possess pharmacological and anticancer activities. The present study was designed to test the hypothesis that AITC suppressed the invasion and migration of epidermal growth factor (EGF)-stimulated HT29 cells and to elucidate the mechanisms for the antimetastatic abilities in vitro. The invasion and migration of EGF-stimulated HT29 cells were determined individually by Transwell cell invasion and wound-healing assays. Our results showed that AITC effectively inhibited both the invasive and migratory ability of HT29 cells. Furthermore, AITC downregulated the protein levels of matrix metalloproteinase-2 (MMP-2), MMP-9 and mitogen-activated protein kinases (MAPKs) (p-JNK, p-ERK and p-p38) by western blot analysis in HT29 cells following EGF induction. Thus, the metastatic responses in AITC-treated HT29 cells after EGF stimulation were mediated by the MMP-2/-9 and MAPK signaling pathways. We also used gene expression microarrays to investigate the gene levels related to cell growth, G-protein coupled receptor, angiogenesis, cell adhesion, cell cycle and mitosis, cell migration, cytoskeleton organization, DNA damage and repair, transcription and translation, EGFR and PKB/mTOR signals. In summary, it is possible that AITC suppresses the invasion and migration of EGF-induced HT29 cells, resulting from MMP-2/-9 and MAPKs. Hence, AITC may be beneficial in the treatment of human colorectal adenocarcinoma in the future.

Stimulatory effect of phenylephrine on the secretion of β-endorphin from rat adrenal medulla in vitro

In an attempt to investigate the role of alpha1-adrenoceptors in the regulation of opioid secretion from adrenal gland, phenylephrine was employed to investigate the effect on secretion of beta-endorphin-like immunoreactivity (BER) from adrenal medulla of rat in vitro. Phenylephrine enhanced the BER from isolated adrenal medulla in a concentration-dependent manner and this action was abolished by the antagonists of alpha1-adrenoceptors, prazosin and tamsulosin. Investigations of signal pathway further support that an activation of alpha1-adrenoceptors is responsible for the stimulatory effect of phenylephrine on BER secretion from adrenal medulla. In the presence of U73312, the specific inhibitor of phospholipase C (PLC), phenylephrine-induced change of BER was reduced in a concentration-dependent manner but it was not affected by U73343, the negative control of U73312. Moreover, chelerythrine and GF 109203X diminished the action of phenylephrine at concentration sufficient to inhibit protein kinase C (PKC). In conclusion, our results suggest that an activation of alpha1-adrenoceptors in adrenal medulla by phenylephrine may enhance the secretion of opioids from adrenal gland of rat via signals of PLC-PKC pathway.