stilbenes and 3-methyladenine

Resveratrol, a natural product, has been revealed to exert antitumor effects in multiple types of tumors. However, the antitumor effects of resveratrol on hepatocellular carcinoma (HCC) and its potential underlying mechanisms have not yet been elucidated. The present study demonstrated that resveratrol inhibited viability, proliferation, invasion and migration of HCC cells significantly in a time‑ and dose‑dependent manner, indicating that resveratrol exerted antitumor effects in HCC. Furthermore, relative expression of autophagy‑related proteins Beclin1 and LC3 II/I ratio was increased while p62 expression was decreased by resveratrol treatment dose‑dependently. The LC3+ puncta formation, which represented autophagosome formation was also markedly dose‑dependently upregulated by resveratrol treatment, suggesting that resveratrol induced autophagy in HCC cells. In addition, treatment with autophagy inhibitor 3‑methyladenine (3‑MA) counteracted the inhibitory effect of resveratrol on HCC cell proliferation, invasion and migration, indicating that suppressing autophagy may hamper the antitumor effect of resveratrol in HCC. It was revealed that resveratrol upregulated the expression of p53 while decreasing the ratio of phosphorylated protein kinase B (p‑Akt)/Akt in HCC cells. Treatment with p53 inhibitor pifithrin‑α and Akt activator insulin‑like growth factor‑1 decreased the expression of Beclin1 while significantly promoting cell proliferation, invasion and migration compared with the resveratrol treatment group. Taken together, the results of the present study revealed that resveratrol inhibited the proliferation and mobility of HCC cells through inducing autophagy via activating p53 and inhibiting phosphoinositide 3‑kinase/Akt. Enhancing autophagy can augment the antitumor effects of resveratrol in HCC. Therefore, combining resveratrol with an autophagy inducer may be a viable option for treating HCC.

Topics: Adenine; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Benzothiazoles; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Humans; Liver Neoplasms; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Resveratrol; Signal Transduction; Stilbenes; Toluene; Tumor Suppressor Protein p53; Up-Regulation

To investigate the effects of resveratrol on autophagy in the chronically diabetic nephropathy and to study the effects of the different expression of microRNAs after resveratrol (RSV) treated in db/db mice (diabetic mice).. Db/m (non- diabetic) and db/db mice were randomly divided into intra gastric RSV treatment group or control group. Renal tissues were prepared for HE/PAS staining. In vitro, mouse podocytes cell lines were grown in different mediums with different dose of resveratrol treatment. microRNA (miRNA) gene chips assay was performed for differentially expressed miRNAs screening. Western blot was used to detect protein levels.. In vivo, RSV significantly decreased urinary albumin, serum creatinine, mesangial area and glomerular size in db/db mice. After RSV treatment, LC3-II/LC3-I and synaptopodin were increased while cleaved-caspase 3 was decreased in kidney tissues. In vitro, podocytes treated with RSV exhibited significantly increased LC3-II/LC3-I and decreased cleaved caspase 3. Moreover, this effect of RSV can be enhanced by rapamycin (RAPA, an activator of autophagy) but partially reversed by 3-MA (an autophagy inhibitor). Further, we found that miR-18a-5p was significantly upregulated after RSV treatment in db/db mice. Overexpression of miR-18a-5p in podocytes resulted in significant inhibition of cleaved-caspase 3 protein, and increased the ratio of LC3-II/LC3-I. Dual luciferase report assay validated that Atactic telangiectasis mutation (ATM) was a target of miR-18a-5p. In podocytes, downregulation of cleaved caspase 3 and the enhanced ratio of protein LC3-II/LC3-I were detected in cells transfected with ATM siRNA.. Role of miRNA-18a-5p in the regulation of autophagy via targeting ATM may represent a promising therapeutic target for preventing and attenuating diabetic nephropathy.

Topics: Adenine; Animals; Ataxia Telangiectasia Mutated Proteins; Autophagy; Caspase 3; Cell Line; Creatinine; Diabetes Mellitus, Experimental; Down-Regulation; Male; Mice; Mice, Obese; MicroRNAs; Microtubule-Associated Proteins; Resveratrol; RNA Interference; RNA, Small Interfering; Sirolimus; Stilbenes; Up-Regulation

Several studies have shown that combination treatment with natural products and chemotherapy agents can improve the sensitivity and cytotoxicity of chemotherapy agents. Resveratrol, a natural product, has many biological effects including antitumor and antiviral activities, as well as vascular protective effect. The aim of this study is to investigate the synergistic anticancer effect of resveratrol in combination with cisplatin and the potential anticancer mechanisms involved in A549 cells. The results obtained from Cell Counting Kit-8 and isobolographic analysis demonstrated that combination of resveratrol and cisplatin resulted in synergistic cytotoxic effects in A549 cells. Results from Hoechst staining, flow cytometry and western blot analysis suggested that resveratrol enhanced cisplatin-mediated apoptosis. Meanwhile, the changes of LC3-II and P62 levels and formation of autophagosome suggested that resveratrol in combination with cisplatin triggered autophagy. More importantly, inhibiting autophagy by 3-methyladenine markedly attenuated the apoptosis caused by combination of resveratrol and cisplatin in A549 cells. Taken together, our study provides the first evidence that resveratrol combined with cisplatin synergistically induce apoptosis via modulating autophagic cell death in A549 cells. These findings also help us to understand the role of natural products in combination with chemotherapy agents in lung cancer.

Topics: A549 Cells; Adenine; Adenocarcinoma, Bronchiolo-Alveolar; Apoptosis; Autophagosomes; Autophagy; Cisplatin; Drug Synergism; Humans; Lung Neoplasms; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Neoplasm Proteins; Proto-Oncogene Proteins c-akt; Resveratrol; RNA-Binding Proteins; Stilbenes

The emergence of multidrug resistance (MDR), meaning that cancer cells develop simultaneous resistance to different drugs, has limited the clinical efficacy and application of chemotherapy. Pterostilbene, a naturally occurring phytoalexin exerts a variety of pharmacologic activities, including cancer prevention, cytotoxicity, and antioxidant activity. In this study, results proved the capability of pterostilbene to effectively inhibit the cell viability of docetaxel-induced MDR human lung cancer cell lines through cell cycle arrest and apoptosis. Meanwhile, the observation of LC3-II production and formation of acidic vesicular organelles revealed an induction of autophagy at an early stage by pterostilbene, which was triggered by an inhibition of the AKT and JNK pathways and activation of ERK1/2. Furthermore, pretreatment with the autophagy inhibitors 3-methyladenine and bafilomycin A1 or with beclin-1 small interfering RNA was able to enhance pterostilbene-triggered apoptosis. In conclusion, this study demonstrated that pterostilbene causes autophagy and apoptosis in lung cancer cells. Furthermore, pterostilbene in combination with autophagy inhibitors may strengthen the efficiency of chemotherapeutic strategies in both chemosensitive and chemoresistant lung cancer cells, which may be of immense value for the clinical management of lung cancer patients with MDR.

Topics: Adenine; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Docetaxel; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; Humans; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; Macrolides; Membrane Proteins; Microtubule-Associated Proteins; Proto-Oncogene Proteins c-akt; RNA Interference; Signal Transduction; Stilbenes; Taxoids; Time Factors; Transfection

Combretastatin A-4 (CA-4) has already entered clinical trials of solid tumors over ten years. However, the limited anticancer activity and dose-dependent toxicity restrict its clinical application. Here, we offered convincing evidence that CA-4 induced autophagy in various cancer cells, which was demonstrated by acridine orange staining of intracellular acidic vesicles, the degradation of p62, the conversion of LC3-I to LC3-II and GFP-LC3 punctate fluorescence. Interestingly, CA-4-mediated apoptotic cell death was further potentiated by pretreatment with autophagy inhibitors (3-methyladenine and bafilomycin A1) or small interfering RNAs against the autophagic genes (Atg5 and Beclin 1). The enhanced anticancer activity of CA-4 and 3-MA was further confirmed in the SGC-7901 xenograft tumor model. These findings suggested that CA-4-elicited autophagic response played a protective role that impeded the eventual cell death while autophagy inhibition was expected to improve chemotherapeutic efficacy of CA-4. Meanwhile, CA-4 treatment led to phosphorylation/activation of JNK and JNK-dependent phosphorylation of Bcl-2. Importantly, JNK inhibitor or JNK siRNA inhibited autophagy but promoted CA-4-induced apoptosis, indicating a key requirement of JNK-Bcl-2 pathway in the activation of autophagy by CA-4. We also identified that pretreatment of Bcl-2 inhibitor (ABT-737) could significantly enhance anticancer activity of CA-4 due to inhibition of autophagy. Taken together, our data suggested that the JNK-Bcl-2 pathway was considered as the critical regulator of CA-4-induced protective autophagy and a potential drug target for chemotherapeutic combination.

Topics: Adenine; Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 5; Beclin-1; Biphenyl Compounds; Cell Line, Tumor; Humans; Macrolides; MAP Kinase Signaling System; Membrane Proteins; Mice; Mice, Nude; Microtubule-Associated Proteins; Nitrophenols; Phosphorylation; Piperazines; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Stilbenes; Sulfonamides; Xenograft Model Antitumor Assays

High expression of vimentin, a canonical mesenchymal marker, is linked with poor prognosis in triple negative breast cancer (TNBC), implying that vimentin may be a potential biomarker in the application of TNBC therapy. Pterostilbene (PTE) has shown anti-invasion activity, and thus, we investigated whether PTE inhibited the epithelial-mesenchymal transition (EMT) in TNBC. Here, we show that PTE decreases the vimentin expression, but that the effect was transient. PTE stimulated Fas signaling, which drives EMT by the ERK1/2 and GSK3β/β-catenin pathways, supporting Fas signaling induction involved in EMT regulation. PTE also triggered autophagy in TNBC. The treatment of TNBC with 3-methyladenine an autophagy inhibitor, not only sustained PTE-inhibited EMT but also significantly promoted anti-proliferation, which indicates that autophagy plays a cyto-protective role and is associated with EMT. Taken together, these data showed that Fas signaling and autophagy accelerated the aggressiveness of TNBC. Inhibition of autophagy or Fas signaling may provide novel targets for TNBC therapy.

Topics: Adenine; Antineoplastic Agents, Phytogenic; Autophagy; beta Catenin; Cell Line, Tumor; Cell Proliferation; Epithelial-Mesenchymal Transition; fas Receptor; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Signal Transduction; Stilbenes; Triple Negative Breast Neoplasms; Vimentin

Inflammation participates centrally in all stages of atherosclerosis (AS), which begins with inflammatory changes in the endothelium, characterized by expression of the adhesion molecules. Resveratrol (RSV) is a naturally occurring phytoalexin that can attenuate endothelial inflammation; however, the exact mechanisms have not been thoroughly elucidated. Autophagy refers to the normal process of cell degradation of proteins and organelles, and is protective against certain inflammatory injuries. Thus, we intended to determine the role of autophagy in the antiinflammatory effects of RSV in human umbilical vein endothelial cells (HUVECs). We found that RSV pretreatment reduced tumor necrosis factor ? (TNF/TNF?)-induced inflammation and increased MAP1LC3B2 (microtubule-associated protein 1 light chain 3 ? 2) expression and SQSTM1/p62 (sequestosome 1) degradation in a concentration-dependent manner. A bafilomycin A 1 (BafA1) challenge resulted in further accumulation of MAP1LC3B2 in HUVECs. Furthermore, autophagy inhibitors 3-methyladenine (3-MA), chloroquine as well as ATG5 and BECN1 siRNA significantly attenuated RSV-induced autophagy, which, subsequently, suppressed the downregulation of RSV-induced inflammatory factors expression. RSV also increased cAMP (cyclic adenosine monophosphate) content, the expression of PRKA (protein kinase A) and SIRT1 (sirtuin 1), as well as the activity of AMPK (AMP-activated protein kinase). RSV-induced autophagy in HUVECs was abolished in the presence of inhibitors of ADCY (adenylyl cyclase, KH7), PRKA (H-89), AMPK (compound C), or SIRT1 (nicotinamide and EX-527), as well as ADCY, PRKA, AMPK, and SIRT1 siRNA transfection, indicating that the effects of RSV on autophagy induction were dependent on cAMP, PRKA, AMPK and SIRT1. In conclusion, RSV attenuates endothelial inflammation by inducing autophagy, and the autophagy in part was mediated through the activation of the cAMP-PRKA-AMPK-SIRT1 signaling pathway.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Autophagy; Carbazoles; Cyclic AMP; Endothelium, Vascular; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Isoquinolines; Resveratrol; Signal Transduction; Sirtuin 1; Stilbenes; Sulfonamides; Tumor Necrosis Factor-alpha; Vasculitis

Angiogenesis plays an important role in many pathological processes. Identification of novel anti-angiogenic agents will provide new insights into the mechanisms for angiogenesis as well as potential lead compounds for developing new drugs. In the present study, a series of resveratrol methylated derivatives have been synthesized and screened. We found trans-3,4-dimethoxystilbene (3,4-DMS) with the fullest potential to develop as an anti-angiogenic agent. In vitro and in vivo analyses suggested that 3,4-DMS could effectively inhibit endothelial cell proliferation, migration, tube formation, and endogenous neovascularization. Our results showed that 3,4-DMS exerted its anti-angiogenic effect likely through induction of endothelial cell apoptosis via a pathway involving p53, Bax, cytochrome c, and caspase proteases. Moreover, 3,4-DMS also induced macroautophagy in endothelial cells through activation of AMPK and the downstream inhibition of mTOR signaling pathway. Further studies indicated that intracellular calcium ([Ca(2+)](i)) might bridge the 3,4-DMS-induced apoptosis and macroautophagy through modulating reactive oxygen species (ROS) levels in endothelial cells. Combination of 3,4-DMS with inhibitor of autophagy, such as 3-methyladenine (3-MA) and autophagy-related gene (ATG) 5 small interfering RNA (siRNA), potentiated the pro-apoptotic and anti-angiogenic effects of 3,4-DMS. Our study provides a novel angiogenic inhibitor and a useful tool in exploring the molecular mechanisms for the crosstalk between apoptosis and macroautophagy in endothelial cells. 3,4-DMS could be served as a potential lead compound for developing a class of new drugs targeting angiogenesis-related diseases.

Topics: Adenine; AMP-Activated Protein Kinases; Angiogenesis Inhibitors; Apoptosis; Autophagy; Autophagy-Related Protein 5; bcl-2-Associated X Protein; Calcium; Caspase 3; Caspase 9; Cell Line; Cell Movement; Cell Proliferation; Cell Survival; Cytochromes c; Human Umbilical Vein Endothelial Cells; Humans; Microtubule-Associated Proteins; Neovascularization, Physiologic; Reactive Oxygen Species; Resveratrol; RNA Interference; RNA, Small Interfering; Stilbenes; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53

Bladder cancer is one of the most common malignancies in the world. The majority of bladder cancer deaths are due to unresectable lesions that are resistant to chemotherapy. Pterostilbene (PT), a naturally occurring phytoalexin, possesses a variety of pharmacologic activities, including antioxidant, cancer prevention activity and cytotoxicity to many cancers. We found that PT effectively inhibits the growth of sensitive and chemoresistant human bladder cancer cells by inducing cell cycle arrest, autophagy and apoptosis. Down-regulations of Cyclin A, B and D1 and pRB are the results of PT-induced cell cycle arrest.. Autophagy occurred at an early stage and was observed through the formation of acidic vesicular organelles (the marker for autophagy) and microtubule-associated protein 1 light chain 3-II production. Apoptosis occurred at a later stage and was detected by Annexin V and 4',6-diamidino-2-phenylindole staining. PT-induced autophagy was triggered by the inhibition of active human protein kinase/the mammalian TOR/p70S6K pathway and activation of extracellular signal-regulated kinase pathway. Inhibition of autophagy by pretreatment with 3-methyladenine, bafilomycin A1, Beclin 1 or extracellular signal-regulated kinase short hairpin RNA enhanced PT-triggered apoptosis.. This is the first study to demonstrate that PT causes autophagy in cancer cells and suggests that PT could serve as a new and promising agent for the treatment of sensitive and chemoresistant bladder cancer cells.

Topics: Adenine; Apoptosis; Autophagy; Cell Cycle; Cell Line, Tumor; Down-Regulation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinases; Signal Transduction; Stilbenes; Urinary Bladder Neoplasms