Chemoprevention of Colorectal Cancer by Artocarpin, a Dietary Phytochemical from Artocarpus heterophyllus.

Artocarpus heterophyllus is an evergreen tree distributed in tropical regions, and its fruit (jackfruit) is well-known as the world's largest tree-borne fruit. Although A. heterophyllus has been widely used in folk medicines against inflammation, its potential in cancer chemoprevention remains unclear. Herein we identified artocarpin from A. heterophyllus as a promising colorectal cancer chemopreventive agent by targeting Akt kinase. Phenotypically, artocarpin exhibited selective cytotoxicity against human colon cancer cells. Artocarpin impaired the anchorage-independent growth capability, suppressed colon cancer cell growth, and induced a G1 phase cell cycle arrest which was followed by apoptotic as well as autophagic cell death. Mechanistic studies revealed that artocarpin directly targeted Akt 1 and 2 kinase activity evidenced by in vitro kinase assay, ex vivo binding assay as well as Akt downstream cellular signal transduction. Importantly, oral administration of artocarpin attenuated colitis-associated colorectal tumorigenesis in mice. Taken together, artocarpin, a bioactive component of A. heterophyllus, might merit investigation as a potential colorectal cancer chemopreventive agent.

Antiplatelet and Antithrombotic Effects of the Extract of Lindera obtusiloba Leaves.

Lindera obtusiloba has been used in traditional herbal medicine for the treatment of blood stasis and inflammation. The leaves of Lindera obtusiloba have been reported to exhibit various physiological activities. However, there is little information available on their antiplatelet and antithrombotic activities. Thus, the present study aimed to evaluate the effect of Lindera obtusiloba leaf extract (LLE) on platelet activities, coagulation and thromboembolism. In a platelet aggregation study, LLE significantly inhibited various agonist-induced platelet aggregations in vitro and ex vivo. Furthermore, LLE significantly inhibited collagen-induced thromboxane A2 (TXA2) production in rat platelets. In addition, oral administration of LLE was protective in a mouse model of pulmonary thromboembolism induced by intravenous injection of a mixture of collagen and epinephrine. Interestingly, LLE did not significantly alter prothrombin time (PT) and activated partial thromboplastin time (aPTT). This study indicates that the antithrombotic effects of LLE might be due to its antiplatelet activities rather than anticoagulation. Taken together, these results suggest that LLE may be a candidate preventive and therapeutic agent in cardiovascular diseases associated with platelet hyperactivity.

Hirsutenone in Alnus extract inhibits akt activity and suppresses prostate cancer cell proliferation.

Although specific compounds found in some East Asian traditional medicines have been shown to exhibit bioactive properties, their molecular mechanisms of action remain elusive. The bark of the Alnus species has been used for the treatment of various pathological conditions including hemorrhage, alcoholism, fever, diarrhea, skin diseases, inflammation, and cancer in East Asia for centuries. In this study, we show that hirsutenone, a bioactive compound in Alnus japonica, exhibits anti-cancer effects against prostate cancer through a direct physical inhibition of Akt1/2. Hirsutenone suppressed anchorage-dependent and independent cell growth of PC3 and LNCaP human prostate cancer cells. Annexin V and Propidium iodide (PI) staining results demonstrated that hirsutenone strongly induces apoptotic cell death in both PC3 and LNCaP cells. Furthermore, treatment of hirsutenone attenuated phosphorylation of mammalian target of rapamycin (mTOR), a downstream substrate of Akt, without affecting Akt phosphorylation. Kinase and pull-down assay results clearly show that hirsutenone inhibits Akt1 and 2 by direct binding in an adenosine triphosphate (ATP)-noncompetitive manner in vitro and ex vivo. Our results show that hirsutenone suppresses human prostate cancer by targeting Akt1 and 2 as a key component to explain for anti-cancer activity of Alnus species.

The ginsenoside 20-O-ß-D-glucopyranosyl-20(S)-protopanaxadiol induces autophagy and apoptosis in human melanoma via AMPK/JNK phosphorylation.

Studies have shown that a major metabolite of the red ginseng ginsenoside Rb1, called 20-O-ß-D-glucopyranosyl-20(S)-protopanaxadiol (GPD), exhibits anticancer properties. However, the chemotherapeutic effects and molecular mechanisms behind GPD action in human melanoma have not been previously investigated. Here we report the anticancer activity of GPD and its mechanism of action in melanoma cells. GPD, but not its parent compound Rb1, inhibited melanoma cell proliferation in a dose-dependent manner. Further investigation revealed that GPD treatment achieved this inhibition through the induction of autophagy and apoptosis, while Rb1 failed to show significant effect at the same concentrations. The inhibitory effect of GPD appears to be mediated through the induction of AMPK and the subsequent attenuation of mTOR phosphorylation. In addition, GPD activated c-Jun by inducing JNK phosphorylation. Our findings suggest that GPD suppresses melanoma growth by inducing autophagic cell death and apoptosis via AMPK/JNK pathway activation. GPD therefore has the potential to be developed as a chemotherapeutic agent for the treatment of human melanoma.

Discovery of the novel mTOR inhibitor and its antitumor activities in vitro and in vivo.

The phosphoinositide 3-kinase (PI3-K)/Akt and mTOR signaling pathway plays a critical role in cell survival and proliferation and is often aberrantly activated in many types of cancer. The mTOR kinase protein, one of the key molecules in this pathway, has been shown to be an important target for cancer therapy. In the present study, a ligand docking method was used to screen for novel scaffold mTOR inhibitors. Sixty thousand compounds in the Natural Product Database were screened against the mTOR homologous structure, and 13 commercially available compounds listed in the top-ranked 100 compounds were selected for further examination. Compound [(E)-3-(4-(benzo[d][1,3]dioxol-5-yl)-2-oxobut-3-en-1-yl)- 3-hydroxyindolin-2-one; designated herein as 3HOI-BA-01] was then selected for further study of its antitumor activity. An in vitro study has shown that 3HOI-BA-01 inhibited mTOR kinase activity in a dose-dependent manner by directly binding with mTOR. In a panel of non-small cell lung cancer cells, the compound also attenuated mTOR downstream signaling, including the phosphorylation of p70S6K, S6, and Akt, resulting in G1 cell-cycle arrest and growth inhibition. Results of an in vivo study have shown that intraperitoneal injection of 3HOI-BA-01 in A549 lung tumor-bearing mice effectively suppressed cancer growth without affecting the body weight of the mice. The expression of downstream signaling molecules in the mTOR pathway in tumor tissues was also reduced after 3HOI-BA-01 treatment. Taken together, we identified 3HOI-BA-01 as a novel and effective mTOR inhibitor.

Identification of an Aurora kinase inhibitor specific for the Aurora B isoform.

Aurora kinases play an important role in chromosome alignment, segregation, and cytokinesis during mitosis. In the present study, we used a ligand docking method to explore the novel scaffold of potential Aurora B inhibitors. One thousand compounds from our in-house compound library were screened against the Aurora B structure and one compound, (E)-3-((E)-4-(benzo[d][1,3]dioxol-5-yl)-2-oxobut-3-en-1-ylidene)indolin-2-one (designated herein as HOI-07) was selected for further study. HOI-07 potently inhibited in vitro Aurora B kinase activity in a dose-dependent manner, without obvious inhibition of another 49 kinases, including Aurora A. This compound suppressed Aurora B kinase activity in lung cancer cells, evidenced by the inhibition of the phosphorylation of histone H3 on Ser10 in a dose- and time-dependent manner. This inhibition resulted in apoptosis induction, G(2)-M arrest, polyploidy cells, and attenuation of cancer cell anchorage-independent growth. Moreover, knocking down the expression of Aurora B effectively reduced the sensitivity of cancer cells to HOI-07. Results of an in vivo xenograft mouse study showed that HOI-07 treatment effectively suppressed the growth of A549 xenografts, without affecting the body weight of mice. The expression of phospho-histone H3, phospho-Aurora B, and Ki-67 was also suppressed in the HOI-07 treatment group. Taken together, we identified HOI-07 as a specific Aurora B inhibitor, which deserves further investigation.

Identification of mammalian target of rapamycin as a direct target of fenretinide both in vitro and in vivo.

N-(4-hydroxyphenyl) retinamide (4HPR, fenretinide) is a synthetic retinoid that has been tested in clinical trials as a cancer therapeutic and chemopreventive agent. Although 4HPR has been shown to be cytotoxic to many kinds of cancer cells, the underlying molecular mechanisms are only partially understood. Until now, no direct cancer-related molecular target has been reported to be involved in the antitumor activities of 4HPR. Herein, we found that 4HPR inhibited mammalian target of rapamycin (mTOR) kinase activity by directly binding with mTOR, which suppressed the activities of both the mTORC1 and the mTORC2 complexes. The predicted binding mode of 4HPR with mTOR was based on a homology computer model, which showed that 4HPR could bind in the ATP-binding pocket of the mTOR protein through hydrogen bonds and hydrophobic interactions. In vitro studies also showed that 4HPR attenuated mTOR downstream signaling in a panel of non-small-cell lung cancer cells, resulting in growth inhibition. Moreover, knockdown of mTOR in cancer cells decreased their sensitivity to 4HPR. Results of an in vivo study demonstrated that i.p. injection of 4HPR in A549 lung tumor-bearing mice effectively suppressed cancer growth. The expression of mTOR downstream signaling molecules in tumor tissues was also decreased after 4HPR treatment. Taken together, our results are the first to identify mTOR as a direct antitumor target of 4HPR both in vitro and in vivo, providing a valuable rationale for guiding the clinical uses of 4HPR.

Simvastatin and lovastatin inhibit breast cell invasion induced by H-Ras.

Breast cancer mortality is strongly related to the invasive and metastatic potential of tumor cells. We previously showed that an active mutant of H-Ras induced invasive phenotype of MCF10A human breast epithelial cells. Membrane anchoring of Ras requires isoprenylation which involves the activity of 3-hydroxy 3-methylglutaryl (HMG)-CoA reductase. In this study, we investigated the inhibitory effect of HMG-CoA reductase inhibitors, widely used for hypercholesterolemia, on H-Ras-induced invasion of MCF10A cells. Treatment of H-Ras MCF10A cells with simvastatin and lovastatin markedly decreased isoprenylated H-Ras in membrane fraction while the unprenylated H-Ras was increased in cytosol fraction, demonstrating that these statins inhibited membrane anchoring of H-Ras in MCF10A cells. Simvastatin and lovastatin significantly inhibited H-Ras-induced invasion which was reversed by farnesyl pyrophosphate (FPP), indicating that the inhibitory effect was related to inhibition of the biosynthesis of prenylated derivatives. Statins downregulated matrix metalloproteinase (MMP)-9 and, to a lesser extent, MMP-2 in H-Ras MCF10A cells. Simvastatin and lovastatin inactivated H-Ras downstream signaling molecules, possibly by inhibiting H-Ras membrane localization and thus its function in MCF10A cells. Taken together, this study clearly demonstrated the inhibitory effect of simvastatin and lovastatin on H-Ras-induced invasion, MMP expression and signal transduction in MCF10A breast epithelial cells, providing supporting rationale for future statin trials as a therapeutic intervention to regulate breast cancer metastasis.