Cordycepin Inhibits Enterovirus A71 Replication and Protects Host Cell from Virus-Induced Cytotoxicity through Adenosine Action Pathway.

Enterovirus A71 (EV-A71) infection typically causes mild illnesses, such as hand-foot-and-mouth disease (HFMD), but occasionally leads to severe or fatal neurological complications in infants and young children. Currently, there is no specific antiviral treatment available for EV-A71 infection. Thus, the development of an effective anti-EV-A71 drug is required urgently. Cordycepin, a major bioactive compound found in Cordyceps fungus, has been reported to possess antiviral activity. However, its specific activity against EV-A71 is unknown. In this study, the potency and role of cordycepin treatment on EV-A71 infection were investigated. Results demonstrated that cordycepin treatment significantly reduced the viral load and viral ribonucleic acid (RNA) level in EV-A71-infected Vero cells. In addition, EV-A71-mediated cytotoxicity was significantly inhibited in the presence of cordycepin in a dose-dependent manner. The protective effect can also be extended to Caco-2 intestinal cells, as evidenced by the higher median tissue culture infectious dose (TCID50) values in the cordycepin-treated groups. Furthermore, cordycepin inhibited EV-A71 replication by acting on the adenosine pathway at the post-infection stage. Taken together, our findings reveal that cordycepin could be a potential antiviral candidate for the treatment of EV-A71 infection.

Tea polyphenol epigallocatechin-3-gallate inhibits cell proliferation in a patient-derived triple-negative breast cancer xenograft mouse model via inhibition of proline-dehydrogenase-induced effects.

Triple-negative breast cancers (TNBCs) lack specific targeted therapy options and have evolved into highly chemo-resistant tumors that metastasize to multiple organs. The present study demonstrated that the proline dehydrogenase (PRODH) mRNA level in paired (tumor vs. normal) human breast tissue samples (n=234) was 6.6-fold greater than normal cells (*p=0.021). We established stable PRODH-overexpressing TNBC (HS578T) cells, and the malignant phenotypes were evaluated using soft agar colony formation and Transwell migration assays. The results demonstrated that PRODH induced epithelial-mesenchymal transition in cancer cells and increased cell proliferation. The present study found that the tea polyphenol epigallocatechin-3-gallate (EGCG) significantly inhibited PRODH and its regulated proteins, such as alpha-smooth muscle actin (alpha-SMA) expression in TNBC cells. These findings support the targeting of the PRODH signaling pathway as a potential therapeutic strategy in preventing cancer cell metastasis. The patient-derived xenograft (PDX) mouse model is highly relevant to real human tumor growth. We established a TNBC-PDX (F4, n=4 in each group)mouse model. The PDX mice were treated with EGCG (50 mg/kg), and the results indicated that EGCG significantly inhibited PDX tumor growth (*p = 0.013). These experiments provide additional evidence to evaluate the antitumor effects of EGCG-induced PRODH inhibition for clinical therapeutic application, especially in TNBC patients.

Study of the antitumor mechanisms of apiole derivatives (AP-02) from Petroselinum crispum through induction of G0/G1 phase cell cycle arrest in human COLO 205 cancer cells.

BACKGROUND: Apiole was isolated from the leaves of various plants and vegetables and has been demonstrated to inhibit human colon cancer cell (COLO 205 cells) growth through induction of G0/G1 cell cycle arrest and apoptotic cell death. This study further explored the antitumor effects of apiole derivatives AP-02, 04, and 05 in COLO 205 cancer cells. METHODS: Human breast (MDA-MB-231, ZR75), lung (A549, PE089), colon (COLO 205, HT 29), and hepatocellular (Hep G2, Hep 3B) cancer cells were treated with apiole and its derivatives in a dose-dependent manner. Flow cytometry analysis was subsequently performed to determine the mechanism of AP-02-induced G0/G1 cell cycle arrest. The in vivo antitumor effect of AP-02 (1 and 5 mg/kg, administered twice per week) was examined by treating athymic nude mice bearing COLO 205 tumor xenografts. The molecular mechanisms of AP-02-induced antitumor effects were determined using western blot analysis. RESULTS: AP-02 was the most effective compound, especially for inhibition of COLO 205 colon cancer cell growth. The cytotoxicity of AP-02 in normal colon epithelial (FHC) cells was significantly lower than that in other normal cells derived from the breast, lung or liver. Flow cytometry analysis indicated that AP-02-induced G0/G1 cell cycle arrest in COLO 205 cells but not in HT 29 cells (< 5 µM for 24 h, **p < 0.01). Tumor growth volume was also significantly inhibited in AP-02 (> 1 mg/kg)-treated athymic nude mice bearing COLO 205 tumor xenografts compared to control mice (*p < 0.05). Furthermore, G0/G1 phase regulatory proteins (p53 and p21/Cip1) and an invasion suppressor protein (E-cadherin) were significantly upregulated, while cyclin D1 was significantly downregulated, in AP-02-treated tumor tissues compared to the control group (> 1 mg/kg, *p < 0.05). CONCLUSIONS: Our results provide in vitro and in vivo molecular evidence of AP-02-induced anti-proliferative effects on colon cancer, indicating that this compound might have potential clinical applications.

The apple polyphenol phloretin inhibits breast cancer cell migration and proliferation via inhibition of signals by type 2 glucose transporter.

Human triple-negative breast cancer (TNBC) is the most aggressive and poorly understood subclass of breast cancer. Glucose transporters (GLUTs) are required for glucose uptake in malignant cancer cells and are ideal targets for cancer therapy. To determine whether the inhibition of GLUTs could be used in TNBC cell therapy, the apple polyphenol phloretin (Ph) was used as a specific antagonist of GLUT2 protein function in human TNBC cells. Interestingly, we found that Ph (10-150 µM, for 24 h) inhibited cell growth and arrested the cell cycle in MDA-MB-231 cells in a p53 mutant-dependent manner, which was confirmed by pre-treatment of the cells with a p53-specific dominant-negative expression vector. We also found that Ph treatment (10-150 µM, for 24 h) significantly decreased the migratory activity of the MDA-MB-231 cells through the inhibition of paxillin/FAK, Src, and alpha smooth muscle actin (α-sMA) and through the activation of E-cadherin. Furthermore, the anti-tumorigenic effect of Ph (10, 50 mg/kg or DMSO twice a week for six weeks) was demonstrated in vivo using BALB/c nude mice bearing MDA-MB-231 tumor xenografts. A decrease in N-cadherin, vimentin and an increase in p53, p21 and E-cadherin were detected in the tumor tissues. In conclusion, inhibition of GLUT2 by the apple polyphenol Ph could potentially suppress TNBC tumor cell growth and metastasis.

An apple a day to prevent cancer formation: Reducing cancer risk with flavonoids.

The purpose of this review is to update and discuss key findings from in vitro and in vivo studies on apple and its biocompounds, with a special focus on its anticancer role. Several studies have proposed that apple and its extracts exhibit a variety of biological functions that may contribute to health benefits including beneficial effects against chronic heart and vascular disorders, respiratory and pulmonary dysfunction, diabetes, obesity, and cancer. In this review, we summarize the molecular mechanism(s) of various components in apple, as established in previous studies that indicated their growth-inhibitory effects in various cancer cell types. Moreover, an attempt is made to delineate the direction of future studies that could lead to the development of apple components as a potent chemopreventive/chemotherapeutic agent against cancer.

Garcinol sensitizes breast cancer cells to Taxol through the suppression of caspase-3/iPLA2 and NF-κB/Twist1 signaling pathways in a mouse 4T1 breast tumor model.

Breast cancer is a significant threat to women's health and has high incidence and mortality. Metastasis in breast cancer patients is a major cause of cancer deaths among women worldwide. Clinical experience suggests that patients with metastatic triple-negative breast cancer (TNBC) relapse quickly and often have chemotherapy resistance. Taxol (paclitaxel) is an effective chemotherapeutic agent for treating metastatic breast cancer, but Taxol at high doses can cause adverse effects and recurrent resistance. Thus, the selection of a synergistic combination therapy is recommended, which is safer and has a more significant response rate than monotherapy. In this study, our strategy is to combine a low dose of Taxol (5 mg kg-1, i.p.) and garcinol (1 mg kg-1, i.g.) to investigate the synergistic antitumor and anti-metastasis effects and to determine the underlying mechanisms of these effects in vivo. For the in vivo study, metastasis-specific mouse mammary carcinoma 4T1 cells were inoculated in Balb/c mice to establish an orthotopic primary tumor and spontaneous metastasis model. Tumor growth and metastases were monitored. The mechanisms of synergistic efficacies were evaluated at different signaling pathways, including proliferation, survival, and epithelial-mesenchymal transition (EMT)-regulated metastatic propensity. We demonstrated that garcinol combined with Taxol significantly increased the therapeutic efficacy when compared with either treatment alone. The synergistic antitumor and anti-metastasis effects were enhanced primarily through the induction of Taxol-stimulated G2/M phase arrest and the inhibition of caspase-3/cytosolic Ca2+-independent phospholipase A2 (iPLA2) and nuclear factor-κB (NF-κB)/Twist-related protein 1 (Twist1) drive downstream events including tumor cell repopulation, survival, inflammation, angiogenesis, invasion, and EMT. Our current findings provide the first experimental evidence that a combination of a low dose of Taxol and garcinol is a promising therapeutic strategy for controlling advanced or metastatic breast cancer. Finally, our results also point to the possible role of NF-κB/Twist1 and caspase-3/iPLA2 signaling pathways as biomarkers to predict the tumor response to treatment.

Pu-erh Tea Extract Attenuates Nicotine-Induced Foam Cell Formation in Primary Cultured Monocytes: An in Vitro Mechanistic Study.

In this study, the mechanisms by which pu-erh tea extract (PETE) attenuates nicotine-induced foam cell formation were investigated. Monocytes were purified from healthy individuals using commercial antibodies coated with magnetic beads. We found that the nicotine-induced (1-10 µM) expression of oxidized low-density lipoprotein receptors (ox-LDLRs) and α9-nAchRs in monocytes was significantly attenuated by 24 h of PETE (10 µg/mL; ∗, p < 0.05) cotreatment. Nicotine (1 µM for 24 h) significantly induced the expression of the surface adhesion molecule ICAM-1 and the monocyte integrin adhesion molecule (CD11b) by human umbilical vein endothelial cells (HUVECs) and triggered monocytes to differentiate into macrophages via interactions with the endothelium. After treatment with nicotine (0.1-10 µM for 24 h), the HUVECs released chemotactic factors (IL-8) to attract monocytes into the tunica intima of the artery, and the monocytes then transformed into foam cells. We demonstrated that PETE treatment (>1 µg/mL for 24 h; ∗, p < 0.05) significantly attenuates nicotine-induced (1 µM) monocyte migration toward HUVECs and foam cell formation. This study suggests that tea components effectively attenuate the initial step (foam cell formation) of nicotine-induced atherosclerosis in circulating monocytes.

P27/Kip1 is responsible for magnolol-induced U373 apoptosis in vitro and in vivo.

Previously, we demonstrated that magnolol, a hydroxylated biphenyl compound isolated from the bark of Magnolia officinalis, at low concentrations (3-10 µM) exerted an antiproliferation effect in colon cancer, hepatoma, and glioblastoma (U373) cell lines through upregulation of the p21/Cip1 protein. Magnolol at a higher concentration of 100 µM, however, induced apoptosis and upregulated p27/Kip1 expression in U373. In the present study, we further studied whether the increased p27/Kip1 expression contributes to the magnolol-induced apoptosis in U373. Our data show that knock-down of p27/Kip1 expression significantly suppressed the magnolol-induced apoptosis, suggesting that p27/Kip1 might play an important role in the regulation of magnolol-induced apoptosis. This notion was further supported by demonstrating that magnolol induced an increase of the caspase activity in U373 in vitro and in vivo, and these effects were abolished by pretransfection of the cell with p27/Kip1 siRNA. To delineate the possible signaling pathways involved in the magnolol-induced increases of p27/Kip1 expression and apoptosis, we found that magnolol (100 µM) increased the levels of phosphorylated cSrc (p-cSrc), p-ERK, p-p38 MAP kinase (p-p38 MAPK), and p-AKT but not p-JNK in U373. Moreover, pretreatment of U373 with a cSrc inhibitor (PP2), a PI3K inhibitor (LY294002), an ERK inhibitor (PD98059), or a p38 MAPK inhibitor (SB203580) but not a JNK inhibitor (SP600125) significantly reduced the magnolol-induced increases of p27/Kip1 protein levels and apoptosis. Taken together, our data suggest that magnolol at a higher concentration of 100 µM induced apopotosis in U373 cells through cSrc-mediated upregulation of p27/Kip1.

In vivo antitumor effects of 4,7-dimethoxy-5-methyl-1,3-benzodioxole isolated from the fruiting body of Antrodia camphorata through activation of the p53-mediated p27/Kip1 signaling pathway.

In this study, 4,7-dimethoxy-5-methyl-1,3-benzodioxole (SY-1) was isolated from three different sources of dried Antrodia camphorata (AC) fruiting bodies. AC is a medicinal mushroom that grows on the inner heartwood wall of Cinnamomum kanehirai Hay (Lauraceae), which is an endemic species that is used in Chinese medicine for its antitumor properties. We demonstrated that SY-1 [given as a 1-30 mg/kg body weight intraperitoneal (ip) injection three times per week] profoundly decreased the growth of COLO-205 human colon cancer cell tumor xenografts in an athymic nude mouse model. We further demonstrated that significant AC extract-mediated antitumor effects were observed at the highest concentration (5 g/kg body weight/day). No gross toxicity signs were observed (i.e., body weight changes, general appearance, or individual organ effects). Frozen COLO-205 xenograft tumors were pulverized in liquid N(2), and the expression of cell cycle regulatory proteins was detected by immunoblotting. We found that the p53-mediated p27/Kip1 protein was significantly induced in the low-dose (1 mg/kg body weight) SY-1-treated tumors, whereas the p21/Cip1 protein levels did not change. The G0/G1 phase cell cycle regulators induced by SY-1 were also associated with a significant decrease in cyclins D1, D3, and A. These results provide further evidence that SY-1 may have significance for cancer chemotherapy.

The in vivo antitumor effects on human COLO 205 cancer cells of the 4,7-dimethoxy-5-(2-propen-1-yl)-1,3-benzodioxole (apiole) derivative of 5-substituted 4,7-dimethoxy-5-methyl-l,3-benzodioxole (SY-1) isolated from the fruiting body of Antrodia camphorate.

CONTEXT: The compound 4,7-dimethoxy-5-(2-propen-1-yl)-1,3-benzodioxole (apiole) has been isolated from several different plant species, including Petroselinum sativum. Our recent study found that apiole is a chemical derivative of 4,7-dimethoxy-5-methyl-l,3-benzodioxole (SY-1), which has been isolated from dried Antrodia camphorata (AC ) fruiting bodies, a traditional Chinese medicine with antitumor properties. AIMS: Our previous in vitro study demonstrated that apiole inhibits the growth of human colon (COLO 205) cancer cells through the arrest of the cell cycle in G0/G1 phase. The in vivo antitumor effects of apiole were evaluated in this study. SETTING AND DESIGN: Apiole was administered to mice at 1-30 mg/kg body weight through intraperitoneal (I.P.) injection three times per week (defined as a dosage of 1×-30×). MATERIALS AND METHODS: The in vivo antitumor effects of apiole were evaluated in mice with xenografts of COLO 205 cells. STATISTICAL ANALYSIS: All of the data are reported as the means ± S.E. Comparisons were performed with a one-way analysis of variance (ANOVA) followed by a Fisher's least significant difference test. Significance was defined as P < 0.05. RESULTS: Apiole (> 1×) markedly decreased the growth of COLO 205 human colon cancer cell tumor xenografts in an athymic nude mouse model system through the up-regulation of cell cycle regulators, such as p53, p21/Cip1, and p27/Kip1. The apiole-induced increase in G0/G1 phase cell cycle regulators was also associated with a significant decrease in the expression of cyclins D1 and D3. Surprisingly, statistically significantly higher tumor volumes were observed in mice that received 5× apiole compared with 30× apiole-treated mice (P < 0.05). No gross signs of toxicity were observed (e.g., body weight changes, general appearance, or individual organ effects) in any group. CONCLUSIONS: Our results show, for the first time, the promising antitumor effects of apiole against colon tumors in an in vivo xenograft model.

Tea polyphenol (-)-epigallocatechin-3-gallate inhibits nicotine- and estrogen-induced α9-nicotinic acetylcholine receptor upregulation in human breast cancer cells.

SCOPE: The aim of this research was to explore whether the tea-polyphenol (-)-epigallocatechin-3-gallate (EGCG) could be used as a potential agent for blocking smoking (nicotine, Nic)- or hormone (estradiol, E2)-induced breast cancer cell proliferation through inhibition of a common signaling pathway. METHODS AND RESULTS: To explore whether Nic (>0.1 µM, 24 h) and E2 (>1 nM, 24 h) significantly increased α9-nicotinic acetylcholine (α9-nicotinic acetylcholine receptor (nAChR)) mRNA and protein expression levels, real-time PCR and immunoblotting analysis experiments were performed in human breast cancer (MCF-7) cells. Luciferase promoter activity experiment was performed to test the α9-nAChR promoter activity affected by Nic, E2 or EGCG. The results indicate that treatment with EGCG (1 µM) profoundly decreases Nic- and E2-induced MCF-7 proliferation by down regulating α9-nAChR expression. The α9-nAChR promoter activity is significantly induced by 24-h treatment with Nic (10 µM) or E2 (10 nM) (>1.8 and ∼2.3-fold, respectively) in MCF-7 cells. Pretreatment with EGCG eliminated the Nic- and E2-induced α9-nAChR promoter-dependent luciferase activity. We further demonstrate that combined treatment with EGCG profoundly inhibits [3H]-Nic/ α9-nAChR binding activity in breast cancer cells. CONCLUSIONS: We found that the EGCG could be used as an agent for blocking smoking (Nic)- or hormone (E2)-induced breast cancer cell proliferation by inhibiting of α9-nAChR signaling pathway. This study reveals the novel antitumor mechanisms of EGCG, and these results may have significant applications for chemopreventive purposes in human breast cancer.

Inhibition of Anchorage-Independent Proliferation and G0/G1 Cell-Cycle Regulation in Human Colorectal Carcinoma Cells by 4,7-Dimethoxy-5-Methyl-l,3-Benzodioxole Isolated from the Fruiting Body of Antrodia camphorate.

In this study, 4,7-dimethoxy-5-methyl-l,3-benzodioxole (SY-1) was isolated from three different sources of dried fruiting bodies of Antrodia camphorate (AC). AC is a medicinal mushroom that grows on the inner heartwood wall of Cinnamomum kanehirai Hay (Lauraceae), an endemic species that is used in Chinese medicine for its anti-tumor and immunomodulatory properties. In this study, we demonstrated that SY-1 profoundly decreased the proliferation of human colon cancer cells (COLO 205) through G0/G1 cell-cycle arrest (50-150 µM) and induction of apoptosis (>150 µM). Cell-cycle arrest induced by SY-1 was associated with a significant increase in levels of p53, p21/Cip1 and p27/Kip1, and a decrease in cyclins D1, D3 and A. In contrast, SY-1 treatment did not induce significant changes in G0/G1 phase cell-cycle regulatory proteins in normal human colonic epithelial cells (FHC). The cells were cultured in soft agar to evaluate anchorage-independent colony formation, and we found that the number of transformed colonies was significantly reduced in the SY-1-treated COLO 205 cells. These findings demonstrate for the first time that SY-1 inhibits human colon cancer cell proliferation through inhibition of cell growth and anchorage-independent colony formation in soft agar. However, the detailed mechanisms of these processes remain unclear and will require further investigation.

Magnolol inhibits human glioblastoma cell proliferation through upregulation of p21/Cip1.

Previously, we demonstrated that magnolol isolated from the bark of Magnolia officinalis has anticancer activity in colon, hepatoma, and leukemia cell lines. In this study, we show that magnolol concentration dependently (0-40 microM) decreased the cell number in a cultured human glioblastoma cancer cell line (U373) and arrested the cells at the G0/G1 phase of the cell cycle. Magnolol treatment decreased the protein levels of cyclins A and D1 and increased p21/Cip1, but not cyclins B and D3, cyclin-dependent kinase (CDK)2, CDK4, CDC25C, Weel, p27/Kip1, and p53. The CDK2-p21/Cip1 complex was increased, and the CDK2 kinase activity was decreased in the magnolol-treated U373. Pretreatment of U373 with p21/Cip1 specific antisense oligodeoxynucleotide prevented the magnolol-induced increase of p21/Cip1 protein levels and the decrease of DNA synthesis. Magnolol at a concentration of 100 microM induced DNA fragmentation in U373. Our findings suggest the potential applications of magnolol in the treatment of human brain cancers.

NF-kappaB-activated tissue transglutaminase is involved in ethanol-induced hepatic injury and the possible role of propolis in preventing fibrogenesis.

The increased expression and cross-linking activity of tissue transglutaminase (tTG) have been demonstrated in acute liver injury and fibrosis. We focused on the molecular mechanisms that contribute to ethanol-induced tTG expression and investigated the efficacy of propolis components in preventing both the tTG expression in vitro and fibrogenesis in vivo. We demonstrate herein that both ERK1/2 and PI3K/Akt pathways can regulate the effects of ethanol on NF-kappaB-dependent transcription and these signaling pathways may be involved in activation of ethanol-mediated tTG expression. We also found that administration of pinocembrin (PIN), one of the major components of propolis, inhibited tTG activation and significantly prevented the development of thioacetamide (TAA)-induced liver cirrhosis. The present study suggests that tTG may be an important member of the cascade of factors necessary for ethanol-induced liver fibrogenesis and PIN could serve as an anti-fibrogenic agent.