lignans and 3-methyladenine

Autophagy is a catabolic process that degrades dysfunctional proteins and organelles and plays critical roles in cancer development. Our preliminary screening identified that extracts of the fruits of Arctium lappa and the fruits of Forsythia suspensa notably suppressed the proliferation of hepatocellular carcinoma HepG2 cells and downregulated the autophagy. In this study, we explored the effect of arctigenin (ARG), a bioactive lignan in both extracts, on cell proliferation and autophagy-related proteins in HepG2 cells. ARG inhibited the proliferation of HepG2 cells. Analysis of autophagy-related proteins demonstrated that ARG might block the autophagy that leads to sequestosome 1/p62 (p62) accumulation. The stage of inhibition in autophagy by ARG differed from those by the autophagy inhibitors 3-methyladenine (3-MA) or chloroquine (CQ). ARG could also inhibit starvation-induced autophagy. Further analysis of apoptosis-related proteins indicated that ARG did not affect caspase-3 activation and PARP cleavage, suggesting that the antiproliferative effect of ARG can occur independently of apoptosis. In summary, our study showed that ARG suppresses cell proliferation and inhibits autophagy, and might lead to the development of agents for autophagy research and cancer chemoprevention.

Topics: Adenine; Apoptosis; Apoptosis Regulatory Proteins; Arctium; Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chloroquine; Forsythia; Furans; Hep G2 Cells; Humans; Lignans; Liver Neoplasms; Sequestosome-1 Protein

Honokiol, an active natural product derived from Magnolia officinalis, exerted anticancer effects through a variety of mechanisms on multiple types of cancers. In this study, the molecular mechanisms of honokiol in suppressing the human oral squamous cell carcinoma (OSCC) cells were evaluated. Treatment of two OSCC cell lines with honokiol resulted in reducing the cell proliferation and arresting the cell cycle at G1 stage which was correlated with the down-regulation of Cdk2 and Cdk4 and the up-regulation of cell cycle suppressors, p21 and p27. In addition, the caspase-dependent programmed cell death was substantially detected, and the autophagy was induced as the autophagosome formation and autophagic flux proceeded. Modulation of autophagy by autophagic inducer, rapamycin or inhibitors, 3-MA or bafilomycin, potentiated the honokiol-mediated anti-OSCC effects where honokiol exerted multiple actions in suppression of MAPK pathway and regulation of Akt/mTOR or AMPK pathways. As compared to clinical therapeutic agent, 5-FU, honokiol exhibited more potent activity against OSCC cells and synergistically enhanced the cytotoxic effect of 5-FU. Furthermore, orally administrated honokiol exerted effective antitumour activity in vivo in OSCC-xenografted mice. Thus, this study revealed that honokiol could be a promising candidate in preventing human OSCCs.

Topics: Adenine; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Biphenyl Compounds; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Fluorouracil; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Lignans; Macrolides; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

To investigate the role of Schisandrin C in odontoblastic differentiation, and its relations between autophagy and mitochondrial biogenesis in human dental pulp cells (HPDCs).. Fresh third molars were used, and cultured for HDPCs. Western blotting technique, Alizarin red S staining, alkaline phosphatase (ALP) activity, and confocal microscopy were used to detect autophagy, mitochondrial biogenesis, and odontoblastic differentiation. To understand the mechanism of Schisandrin C, the HDPCs were treated with lipopolysaccharide (LPS), autophagy and heme oxygenase-1 (HO-1) inhibitors: 3-Methyladenine (3-MA) and Zinc protoporphyrin IX (ZnPP), respectively.. LPS decreased the expression of autophagy molecules [autophagy protein 5 (ATG-5), beclin-1, and microtubule-associated protein 1A/1B light chain 3 (LC3-I/II)] and mitochondrial biogenesis molecules [heme oxygenase-1 (HO-1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)], and disrupted odontoblastic differentiation. The down-regulation of autophagy and mitochondrial biogenesis with 3-MA and ZnPP inhibited odontoblastic differentiation. However, Schisandrin C restored the expression of all the above molecules, even with LPS and inhibitor treatment. This result demonstrates that autophagy and mitochondrial biogenesis plays an essential role in odontoblastic differentiation, and Schisandrin C activates these systems to promote odontoblastic differentiation of HDPCs.. Schisandrin C has potential characters to regulate odontoblastic differentiation, and may be recommended for use as a compound for pulp homeostasis.

Topics: Adenine; Anti-Inflammatory Agents; Autophagy; Autophagy-Related Protein 5; Beclin-1; Cell Differentiation; Cells, Cultured; Cyclooctanes; Dental Pulp; Down-Regulation; Heme Oxygenase-1; Humans; Lignans; Lipopolysaccharides; Microtubule-Associated Proteins; Mitochondria; Molar, Third; Odontoblasts; Organelle Biogenesis; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Polycyclic Compounds; Protoporphyrins

Honokiol, an active constituent extracted from the bark of Magnolia officinalis, possesses anticancer effects. Apoptosis is classified as type I programmed cell death, while autophagy is type II programmed cell death. We previously proved that honokiol induces cell cycle arrest and apoptosis of U87 MG glioma cells. Subsequently in this study, we evaluated the effect of honokiol on autophagy of glioma cells and examined the molecular mechanisms. Administration of honokiol to mice with an intracranial glioma increased expressions of cleaved caspase 3 and light chain 3 (LC3)-II. Exposure of U87 MG cells to honokiol also induced autophagy in concentration- and time-dependent manners. Results from the addition of 3-methyladenine, an autophagy inhibitor, and rapamycin, an autophagy inducer confirmed that honokiol-induced autophagy contributed to cell death. Honokiol decreased protein levels of PI3K, phosphorylated (p)-Akt, and p-mammalian target of rapamycin (mTOR) in vitro and in vivo. Pretreatment with a p53 inhibitor or transfection with p53 small interfering (si)RNA suppressed honokiol-induced autophagy by reversing downregulation of p-Akt and p-mTOR expressions. In addition, honokiol caused generation of reactive oxygen species (ROS), which was suppressed by the antioxidant, vitamin C. Vitamin C also inhibited honokiol-induced autophagic and apoptotic cell death. Concurrently, honokiol-induced alterations in levels of p-p53, p53, p-Akt, and p-mTOR were attenuated following vitamin C administration. Taken together, our data indicated that honokiol induced ROS-mediated autophagic cell death through regulating the p53/PI3K/Akt/mTOR signaling pathway.

Topics: Adenine; Animals; Apoptosis; Ascorbic Acid; Autophagy; Biphenyl Compounds; Caspase 3; Cell Line, Tumor; Dose-Response Relationship, Drug; Down-Regulation; Glioma; Lignans; Mice; Microtubule-Associated Proteins; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53

Our previous reports showed that justicidin A (JA), a novel and pure arylnaphthalide lignan isolated from Justicia procumbens, induces apoptosis of human colorectal cancer cells and hepatocellular carcinoma cells, leading to the suppression of both tumor cell growth in NOD-SCID mice. Here, we reveal that JA induces autophagy in human colorectal cancer HT-29 cells by conversion of autophagic marker LC3-I to LC3-II. Furthermore, LC3 puncta and autophagic vesicle formation, and SQSTM1/p62 suppression were observed. Administration of autophagy inhibitor (bafilomycin A1 and chloroquine) and transfection of a tandem fluorescent-tagged LC3 (mRFP-GFP) reporter plasmid (ptfLC3) demonstrated that JA induces autophagy flux in HT-29 cells. Expression of LC3, SQSTM1, Beclin 1, and nuclear DNA double-strand breaks (representing apoptosis) were also detected in the tumor tissue of HT-29 cells transplanted into NOD-SCID mice orally administrated with JA. In addition, the expression of autophagy signaling pathway-related molecules p-PDK1, p-mTOR, p-p70S6k/p-RPS6KB2 was decreased, whereas that of class III PI3K, Beclin 1, Atg5-Atg12, and mitochondrial BNIP3 was increased in response to JA. Pre-treatment of the cells with class III PI3K inhibitor 3-methyladenine or Atg5 shRNA attenuated JA-induced LC3-II expression and LC3 puncta formation, indicating the involvement of class III PI3K and Atg5. A novel mechanism was demonstrated in the anticancer compound JA; pre-treatment with 3-methyladenine or Atg5 shRNA blocked JA-induced suppression in cell growth and colony formation, respectively, via inhibition of apoptosis. In contrast, administration of apoptosis inhibitor Z-VAD did not affect JA-induced autophagy. Our data suggest the chemotherapeutic potential of JA for treatment of human colorectal cancer.

Topics: Adenine; Animals; Apoptosis; Autophagy; Autophagy-Related Protein 5; Colorectal Neoplasms; Dioxolanes; HT29 Cells; Humans; Lignans; Mice; Mice, SCID; Microtubule-Associated Proteins; Phosphatidylinositol 3-Kinases; RNA, Small Interfering; Signal Transduction

SMXZF is a combination of Rb1, Rg1, schizandrin, and DT-13 (6:9:5:4) derived from Sheng-mai San, a widely used Chinese traditional medicine for the treatment of cardiovascular and cerebral diseases. The present study explores the inhibitory effects and signaling pathways of SMXZF on autophagy induced by cerebral ischemia-reperfusion injury. Male C57BL/6 mice were subjected to ischemia-reperfusion insult by right middle cerebral artery occlusion (MCAO) for 1 hr with subsequent 24 hr reperfusion. Three doses of SMXZF (4.5, 9, and 18 mg/kg) were administered intraperitoneally (i.p.) after ischemia for 1 hr. An autophagic inhibitor, 3-methyladenine (3-MA; 300 μg/kg), was administered i.p. 20 min before ischemia as a positive drug. We found that SMXZF significantly increased cerebral blood flow and reduced the infarct volume, brain water content, and the neurological deficits in a dose-dependent manner. Similar to the positive control, SMXZF at 18 mg/kg also significantly inhibited autophagosome formation. Immunofluorescence staining and Western blotting demonstrated that SMXZF could significantly decrease the expression levels of beclin1 and microtubule-associated protein 1 light chain 3. SMXZF also remarkably inhibited the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) as well as the expression of c-Jun N-terminal kinase (JNK) and its phosphorylation induced by 24 hr reperfusion. Finally, we demonstrated that the optimal administration time of SMXZF was at the early period of reperfusion. This study reveals that SMXZF displays neuroprotective effect against focal ischemia-reperfusion injury, possibly associated with autophagy inactivation through AMPK/mTOR and JNK pathways.

Topics: Adenine; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Autophagy; Brain; Brain Infarction; Cyclooctanes; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Combinations; Drugs, Chinese Herbal; Ginsenosides; Infarction, Middle Cerebral Artery; Lignans; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Polycyclic Compounds; Renal Circulation; Reperfusion Injury; Saponins; Time Factors; TOR Serine-Threonine Kinases