CUR5g, a novel autophagy inhibitor, exhibits potent synergistic anticancer effects with cisplatin against non-small-cell lung cancer.

Autophagy, a highly conserved degradation process of eukaryotic cells, has been proven to be closely related to chemoresistance and metastasis of non-small-cell lung cancer (NSCLC). Autophagy inhibitors, such as chloroquine (CQ) and its derivative hydroxychloroquine (HCQ), has been shown to mediate anticancer effects in preclinical models, especially when combined with chemotherapy. However, the vast majority of autophagy inhibitors, including CQ and HCQ, actually disrupt lysosomal or/and possibly non-lysosomal processes other than autophagy. It is therefore of great significance to discover more specific autophagy inhibitors. In this study, after screening a series of curcumin derivatives synthesized in our laboratory, we found that (3E,5E)-1-methyl-3-(4-hydroxybenzylidene)-5-(3-indolymethylene)-piperidine-4-one (CUR5g) selectively inhibited autophagosome degradation in cancer cells by blocking autophagosome-lysosome fusion. CUR5g did not affect the lysosomal pH and proteolytic function, nor did it disturb cytoskeleton. CUR5g blocked the recruitment of STX17, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, to autophagosomes via a UVRAG-dependent mechanism, resulting in the inability of autophagosomes to fuse with lysosomes. CUR5g alone did not induce apoptosis and necrosis of A549 cells, but significantly inhibited the mobility and colony formation of A549 cells. More excitingly, CUR5g showed no obvious toxicity to normal HUVECs in vitro or mice in vivo. CUR5g enhances the cisplatin sensitivity of A549 cells and effectively inhibited autophagy in tumor tissues in vivo. Collectively, our study identified a new late-stage autophagy inhibitor and provided a novel option for NSCLC treatment, particular when combined with cisplatin.

Identification of Compound CB-2 as a Novel Late-Stage Autophagy Inhibitor Exhibits Inhibitory Potency against A549 Cells.

Autophagy has been recognized as a stress tolerance mechanism that maintains cell viability, which contributes to tumor progression, dormancy, and treatment resistance. The inhibition of autophagy in cancer has the potential to improve the therapeutic efficacy. It is therefore of great significance to search for new autophagy inhibitors. In the present study, after screening a series of curcumin derivatives synthesized in our laboratory, (E)-3-((E)-4-chlorobenzylidene)-5-((5-methoxy-1H-indol-3-yl)methylene)-1-methylpiperidin-4-one (CB-2) was selected as a candidate for further study. We found that CB-2 increased the LC3B-II and SQSTM1 levels associated with the accumulation of autophagosomes in non-small cell lung cancer (NSCLC) A549 cells. The increased level of LC3B-II induced by CB-2 was neither eliminated when autophagy initiation was suppressed by wortmannin nor further increased when autophagosome degradation was inhibited by chloroquine (CQ). CB-2 enhanced the accumulation of LC3B-II under starvation conditions. Further studies revealed that CB-2 did not affect the levels of the key proteins involved in autophagy induction but significantly blocked the fusion of autophagosomes with lysosomes. High-dose CB-2 induced the apoptosis and necrosis of A549 cells, while a lower dose of CB-2 mainly impaired the migrative capacity of A549 cells, which only slightly induced cell apoptosis. CB-2 increased the levels of mitochondrial-derived reactive oxygen species (ROS) while decreasing the mitochondrial membrane potential (MMP). Scavenging ROS via N-acetylcysteine (NAC) reversed CB-2-induced autophagy inhibition and its inhibitory effect against A549 cells. In conclusion, CB-2 serves as a new late-stage autophagy inhibitor, which has a strong inhibitory potency against A549 cells.

Antiproliferative effect and autophagy induction of curcumin derivative ZYX02-Na on the human lung cancer cells A549.

At present, a large number of curcumin derivatives had been produced and identified aiming to replace the curcumin in view of its low bioavailability and stability. Here, a novel curcumin derivative ZYX02-Na was first used to reduce the cell viability of human non-small cell lung cells A549, which was confirmed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry and Western blot analysis showed that ZYX02-Na could lead to cell cycle arrest in G0/G1 phase, which demonstrated that ZYX02-Na inhibited the proliferation of A549 cells. Furthermore, the AMPK/mTOR/4E-BP1 signaling pathway was activated in ZYX02-Na-treated A549 cells. Besides, wounding healing and transwell experiments showed that ZYX02-Na could also inhibited the migration ability of A549 cells. Moreover, we also found that ZYX02-Na could induce autophagy of A549 cells by acridine orange staining, GFP-LC3 subcellular localization observation and Western blotting analysis, respectively. In short, our current studies indicated that ZYX02-Na possessed the antiproliferation effect and autophagy induction on A549 cells, while in vivo anticancer study of ZYX02-Na needs to be done in future.

One novel curcumin derivative ZYX01 induces autophagy of human non-small lung cancer cells A549 through AMPK/ULK1/Beclin-1 signaling pathway.

Presently, curcumin derivatives had been paid more attention in view of their high bioavailability or water solubility, which herein possibly replaced the curcumin for their functional applications in future. Here, one novel chemically synthesized curcumin derivative, ZYX01, was used to identify anti-proliferation activity of human non-small lung cancer cells A549 and its anti-proliferative mechanism. Our study showed that ZYX01 could induce autophagic death of A549 cells by morphological observation, MTT assay, acridine orange staining and MDC assay, which possess a dose-and time-dependent manner. ZYX01-treated A549 cells possessed an increase in LC3-II/LC3-I ratio, upregulation of beclin-1 and downregulation of p62 expression. We further confirmed the cellular AMPK/ULK1/Beclin-1 signaling pathway in A549 cells after ZYX01 treatment. The anti-migration effect of ZYX01 in A549 cells was also explored by wound healing assay and transwell experiment. Current results had confirmed that ZYX01 induced A549 cells autophagy through AMPK/ULK1/Beclin-1 pathway and shed light on the future study on the anti-cancer molecular mechanism.

Identification of compound CA-5f as a novel late-stage autophagy inhibitor with potent anti-tumor effect against non-small cell lung cancer.

Currently, particular focus is placed on the implication of autophagy in a variety of human diseases, including cancer. Discovery of small-molecule modulators of autophagy as well as their potential use as anti-cancer therapeutic agents would be of great significance. To this end, a series of curcumin analogs previously synthesized in our laboratory were screened. Among these compounds, (3E,5E)-3-(3,4-dimethoxybenzylidene)-5-[(1H-indol-3-yl)methylene]-1-methylpiperidin-4-one (CA-5f) was identified as a potent late-stage macroautophagy/autophagy inhibitor via inhibiting autophagosome-lysosome fusion. We found that CA-5f neither impaired the hydrolytic function nor the quantity of lysosomes. Use of an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic screen in combination with bioinformatics analysis suggested that treatment of human umbilical vein endothelial cells (HUVECs) with CA-5f for 1 h suppressed the levels of cytoskeletal proteins and membrane traffic proteins. Subsequent studies showed that CA-5f exhibited strong cytotoxicity against A549 non-small cell lung cancer (NSCLC) cells, but low cytotoxicity to normal human umbilical vein endothelial cells (HUVECs), by increasing mitochondrial-derived reactive oxygen species (ROS) production. Moreover, CA-5f effectively suppressed the growth of A549 lung cancer xenograft as a single agent with an excellent tolerance in vivo. Results from western blot, immunofluorescence, and TdT-mediated dUTP nick end labeling (TUNEL) assays showed that CA-5f inhibited autophagic flux, induced apoptosis, and did not affect the level of CTSB (cathepsin B) and CTSD (cathepsin D) in vivo, which were consistent with the in vitro data. Collectively, these results demonstrated that CA-5f is a novel late-stage autophagy inhibitor with potential clinical application for NSCLC therapy. Abbreviations: 3-MA, 3-methyladenine; ANXA5, annexin A5; ATG, autophagy related; CA-5f, (3E,5E)-3-(3,4-dimethoxybenzylidene)-5-[(1H-indol-3-yl)methylene]-1-methylpiperidin-4-one; CQ, chloroquine; CTSB, cathepsin B; CTSD, cathepsin D; DMSO, dimethyl sulfoxide; DNM2, dynamin 2; EBSS, Earle's balanced salt solution; GFP, green fluorescent protein; HCQ, hydroxyl CQ; HEK293, human embryonic kidney 293; HUVEC, human umbilical vein endothelial cells; LAMP1, lysosomal associated membrane protein 1; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; LDH, lactic acid dehydrogenase; LMO7, LIM domain 7; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; NAC, N-acetyl cysteine; MYO1E, myosin IE; NSCLC, non-small cell lung cancer; PARP1, poly(ADP-ribose) polymerase 1; PI, propidium iodide; RFP, red fluorescent protein; ROS, reactive oxygen species; SQSTM1, sequestosome 1; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Autophagy induction and antiproliferative effect of a novel curcumin derivative MOMI-1 on the human lung cancer cells A549.

To date, there are some chemically synthesized curcumin derivatives which were produced and identified to evade the disadvantages of physiochemical stability and solubility of curcumin. Here, one novel curcumin derivative, (2-(3-{(1E)-{(E)-3-(4-hydroxy-3-methoxybenzylidene)-2-oxocyclohexylidene)methyl)-1H-indol-1-yl)acetic acid}, (abbreviated as MOMI-1) was first used to detect the antiproliferation activity with MTT assays in different cancer cells including A549 lung cancer cells, MCF-7, and HEPG2 cell lines, and exhibited its wide inhibition spectrum. Next, we found that MOMI-1 could induce autophagic genesis of A549 cells by acridine orange or monodansylcadaverine (MDC) staining and green fluorescent protein-light chain 3 (GFP-LC3) recombinant plasmid transfection analysis, respectively. Western blot analysis confirmed the LC3-I/II conversion, beclin-1 increase and p62 reduction of A549 cells after exposure of MOMI-1, which suggested the typical autophagy induction. The following cell cycle test showed that MOMI-1 could block A549 cells in G0/G1 phase. Furthermore, wounding healing experiment and transwell assays demonstrated that MOMI-1 also possessed the antimigration ability of A549 cells. Our current results confirmed that MOMI-1 could inhibit the proliferation and induce autophagy of A549 cells, which provide a new potential chemical candidate of antigrowth of A549 lung cancer cells. Future work needs to focus on the mechanism of autophagy pathway of A549 cells.

A novel synthetic curcumin derivative MHMM-41 induces ROS-mediated apoptosis and migration blocking of human lung cancer cells A549.

Many curcumin derivatives were produced and characterized to improve the physiochemical instability and low solubility of curcumin. Here, MHMM-41 (a novel curcumin derivative) was used to treat non-small lung cancer cells of human (known as A549) and to identify its anti-proliferative activities. Our results suggested that MHMM-41 display no significant cytotoxicity toward normal human lung fibroblast 2BS cells and mouse embryonal fibroblast 3T3 cells. It also had better anti-proliferative activity than curcumin in A549 cells. Further study showed a significant increase of apoptotic A549 cells in time and dose dependent manners. The activation of caspase-3, 8, 9, 12, Bax and PARP proteins were detected. Consequently, MHMM-41 treatment led to the reduction of mitochondrial membrane potential by JC-1 staining and characteristic nuclei fragmentation by Hoechst 33,342 staining, respectively, which showed that A549 apoptosis could be triggered by the extrinsic and intrinsic mitochondrial pathways. The release of ROS was also measured by flow cytometry. Further, wound healing assay and transwell experiments confirmed the anti-migration ability of MHMM-41 in A549 cells. Our current study suggested the potentials of MHMM-41 to inhibit the A549 cell proliferation. However, the intensive mechanical research on the anti-proliferation of A549 cells needs to be performed in the future.

The Interplay between Autophagy and Apoptosis Induced by One Synthetic Curcumin Derivative Hydrazinobenzoylcurcumin in A549 Lung Cancer Cells.

The two important cell autonomic responses, autophagy, and apoptosis, play critical roles in cellular homeostasis and survival. By studying of the synthetic curcumin derivative hydrazinobenzoylcurcumin (HBC), we revealed that it could induce autophagy in nonsmall lung cancer cells (A549). Here, we use the Hoechst 33342 staining, Annexin V/propyliodide double dyeing and Western blotting analysis of PARP protein to demonstrate that HBC could also induce apoptosis in A549 cells. Apoptosis inhibitor (Z-VAD-FMK, 10 µM) treatment helps to promote the cells survival. Moreover, inhibition of apoptosis-promoted HBC-induced autophagy of A549 cells by morphological detection and Western blotting analysis (vice versa). These data indicate that there exist some interconnections between the autophagy and apoptosis induced by HBC. The following work will be carried out to characterize the specific regulation processes between the two cell pathways in A549 cells.

Silver-catalyzed radical tandem cyclization: an approach to direct synthesis of 3-acyl-4-arylquinolin-2(1H)-ones.

A silver-catalyzed efficient and practical synthesis of 3-acyl-4-arylquinolin-2(1H)-ones or 3-acyl-4-aryldihydroquinolin-2(1H)-ones through intermolecular radical addition/cyclization in aqueous solution is reported. This method provides a novel, highly efficient, and straightforward route to substituted quinolin-2-ones or 3,4-dihydroquinolin-2-ones in one step. A possible mechanism for the formation of quinolin-2-ones is proposed.

Novel curcumin analogue IHCH exhibits potent anti­proliferative effects by inducing autophagy in A549 lung cancer cells.

Curcumin is a natural polyphenolic compound that exhibits strong antioxidant and anticancer activities; however, low bioavailability has restricted its application in chemotherapeutic trials. The present study aimed to investigate the anticancer effect of the novel curcumin derivative 2E,6E­2­(1H­indol­3­yl) methylene)­6­(4­hydroxy­3­methoxy benzylidene)­cyclohexanone (IHCH) on A549 lung cancer cells. Cells were treated with IHCH at different concentrations (1­40 µM) for different time periods (1­36 h). Microscopic analysis revealed that IHCH inhibited A549 cell growth and induced the formation of characteristic autophagolysosomes in a dose­ and time­dependent manner. Furthermore, the inhibitory rate of IHCH (40 µM) on A549 cell viability was 77.34% after 36 h of treatment. Acridine orange staining revealed an increase in autophagic vacuoles in the IHCH­treated A549 cells. Monodansylcadaverine staining was used to analyze autophagy rate. Immunocytochemistry revealed an increase in light chain (LC) 3 protein expression in the IHCH­treated cells and western blot analysis detected the conversion of LC3­I to LC3­II, as well as the recruitment of LC3 to autophagosomes in the cytoplasmatic compartment, suggesting the occurrence of autophagy. These findings show that IHCH induced autophagy in A549 cells, which is a novel cell death mechanism induced by curcumin derivatives.

A synthetic curcumin derivative hydrazinobenzoylcurcumin induces autophagy in A549 lung cancer cells.

CONTEXT: Curcumin exhibits growth-suppressive activity against a variety of cancer cells, but low bioavailability restricts its application in chemotherapeutic trials. Nowadays, a growing number of curcumin derivatives or analogs are known, hoping to replace curcumin and circumvent this problem. Hydrazinobenzoylcurcumin (HBC) has been synthesized and identified as a potent inhibitor of cell proliferation in previous reports. OBJECTIVE: This study presents a novel mechanism of cell autophagy induced by HBC in the human non-small lung epithelial carcinoma (A549) cells. MATERIALS AND METHODS: Cells were cultured and treated with HBC at different concentrations (10-80 µM) and at different time periods (1-24 h). Microscopic analysis was used to detect the morphology changes and autophagolysosomes of A549 cells. An acridine orange staining assay was conducted to evaluate the autophagolysosomes and autophagic vacuoles was analyzed by monodansylcadaverine (MDC) and GFP-LC3 transfection analysis. Western blotting was used to assess the conversion of microtubule-associated protein light chain 3 (LC3). RESULTS: HBC could induce A549 cells autophagolysosomes formation in a dose and time-dependent manner and the inhibitory rate of HBC (80 µM) on the viability of A549 cells reached 76.68 ± 5.81% after 24 h of treatment. Autophagic vacuoles increased in a concentration-dependent manner in HBC-treated cell. Furthermore, conversion of LC3-I to LC3-II, accumulation of GFP-tagged LC3 positive intracellular vacuoles and increased fusion of autophagosomes with lysosomes suggested the occurrence of autophagy. CONCLUSION: Our data indicate that HBC induced A549 cell autophagy, which is a novel cell death mechanism induced by curcumin derivatives.

5-Hydr-oxy-1,7-bis-(1H-indol-3-yl)hepta-1,4,6-trien-3-one hemihydrate.

The title compound, C(23)H(18)N(2)O(2)·0.5H(2)O, a derivative of the biologically active compound curcumin, crystallizes with two organic mol-ecules and a solvent water mol-ecule in the asymmetric unit. Each of the two independent mol-ecules is close to being planar (the dihedral angles between the indole ring systems are approximately 9 and 12°) and each exists in the keto-enol form. There is an intra-molecular O-H⋯O hydrogen bond between the keto and enol groups. In the crystal, the components interact by way of N-H⋯N, N-H⋯O and O-H⋯O hydrogen bonds.

Parallel solution-phase synthesis of 4H-benzo[1,4]thiazin-3-one and 1,1-dioxo-1,4-dihydro-2H-1lambda6-benzo[1,4]thiazin-3-one derivatives from 1,5-difluoro-2,4-dinitrobenzene.

This paper discusses the synthesis of privileged structures 4H-benzo[1,4]thiazin-3-one and 1,1-dioxo-1,4-dihydro-2H-1lambda6-benzo[1,4]thiazin-3-one derivatives in a parallel solution-phase manner using 1,5-difluoro-2,4-dinitrobenzene. Each scaffold possesses four diversity points. A cheap and efficient oxidant, urea-hydrogen peroxide (UHP), was applied for the introduction of the sulfone group. The intramolecular cyclization to 1,1-dioxo-1,4-dihydro-2H-1lambda6-benzo[1,4]thiazin-3-one was achieved by microwave assistance or the use of an inorganic base.

[Study on UV-Vis absorption spectra of tetra-azo-aromaticoxy substituted metallophthalocyanines].

UV-Vis absorption spectras of six series (18 kinds) of tetra- azo-aromaticoxy substituted metallophthalocyanines (R4 PcM, R = 4-pyridyloxy, 8-quinolinoxy, 2-methyl-8-quinolinoxy; substitution position: a position and beta position; M = Ni (II), Cu(II), Zn(II)) were measured. The effects of central mentals, the kinds and the positions of substitution groups, and solvents on the metallophthalocyanines' lamdamax in Q-band were discussed. Experimental data show: The lamdamax in Q-band of title complexes is about 680 nm. In contrast with substitution-free metallophthalocyanines(669-671 nm), the lamdamax in Q-band of the title complexes with the same central metal exhibits a different red-shift. The effect of substitution group's kinds on lamdamax in Q-band of the title complexes is more obvious in a position than in beta position, and with the same substitution group and central metal, lamdamax in Q-band of alpha position substituted complexes exhibits more obvious red-shift than beta position substituted complexes. The effects of central metal and solvent on lamda,ax in Q-band of the title complexes aren't obvious.

Parallel approach for solution-phase synthesis of 2-quinoxalinol analogues and their inhibition of LPS-induced TNF-alpha release on mouse macrophages in vitro.

A parallel solution-phase synthesis of 2-quinoxalinol analogues is described. The key step-simultaneous reductions of m-Ar(NO2)2 to m-Ar(NH2)2 was investigated extensively. We obtained preliminary pharmacological activity of those analogues for the inhibition of LPS-induced TNF-alpha release on mouse macrophage in vitro. Two compounds revealed inhibitory activity, with IC50 values of 0.40 microM (7-amino-6-[(3-methoxypropyl)amino]-3-methyl-2-quinoxalinol) and 2.2 microM (7-amino-6-[(3-butoxypropyl)amino]-3-methyl-2-quinoxalinol), respectively.