| Background and objection:Microtubules are components of cytoskeleton and are present in virtually all eukaryotic cells. Microtubules form spindle, centriole, flagllum and nerviduct with other proteins in the cells. Under normal condition, there is a balance between microtubule polymerization and depolymerization and the dynamics regulates the mitosis. Microtubules are involved in many cellular processes, including maintenance of cell shape, cell migration, mitosis, intracellular transport and cell signaling. In the eukaryotic cell cycle, microtubules are polymerized and form the mitotic spindle in prophase, which then moves the chromosomes to the opposite sides of the cell, in preparation for cell division into two daughter cells. Because of this important role in cell proliferation, microtubules have been recognized as one of the successful and efficacious drug targets for the development of novel anti-cancer chemotherapeutics. Microtubule-inhibiting agents (MIAs) currently have been used in clinic therapies work through the suppression of the microtubule dynamics by misdirecting the formation of a functional mitotic spindle in fast dividing tumor cells. This arrests the cells in M phase, thereby leading to apoptosis of the tumor cells.Based on their mechanism of action, Microtubule-inhibiting agents (MIAs) are classified into two broad categories:①microtubule destabilizing agents, including vinca alkaloids and colchicine;②microtubule stabilizing agents, including paclitaxel, epothilone and its analogues. According to their different binding sites on microtubule protein, Microtubule-inhibiting agents (MIAs) are further classified into three groups:colchicine site-binding agents, vinblastine site-binding agents, and paclitaxel site-binding agents. Due to the potent anti-cancer activity, these apoptotic therapeutic agents that target microtubules, including paclitaxel and vinblastine, are among the most commonly prescribed antitumor agents. Paclitaxel is currently recommended as first-line agent in the chemotherapy of lung cancer, breast cancer and ovarian cancer. However, as with other anticancer drugs, intolerable toxicities and the emergence of drug resistance have limited the clinical use of the drugs targeting microtubules. Therefore, a need still exists for discovery and development of novel chemotherapeutic agents that target microtubules, but that show better activicy, lower drug resistance and fewer toxic side effects.The small compound dubbed HA14-1, identified by one of my supervisor--Dr. Huang ziwei, was the first reported Bcl-2inhibitor which can potently induce apoptosis in a wide variety of human cancers, including breast cancer, colorectal cancer, kidney cancer, cervical cancer, lung cancer, brain glioma and leukemia. In subsequent studies, we found that it not only can induce reactive oxygen species (ROS) generation, cytochrome c release, and Caspase-9/-3activation, but it also can bind microtubules in a manner that is competitive with colchicine when its concentration is greater than10μM. In the present study, we used HA14-1as the initial template compound and developed a new class of novel microtubule-targeting agents (2-amino-4-phenyl-4H-chromene-3-carboxylate analogues) named mHAl-19. mHA1,6and11showed more potent and stable than others, so we choosed them to do subsequent biological assessment. After treatment with these agents, morphological changes including cell elongation, asymmetry, and formation of long pseudopodia were observed which are quite different from the apoptotic morphological changes induced by HA14-1treatment, including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation and apoptotic body. This phenomenon indicated that these mHA analogues worked through different pathway from HA14-1to kill cancer cells. Microtubule as the important component of cell shape maintenance was the most probable target. Although the high toxicity of colchicine has prevented its clinical use in cancer therapy, the colchicine-binding site is still a potential drug target that has attracted much attention for development of new agents that bind to this site. Several such compounds have entered clinical trials. Study and find the action mechanism of these mHA analogues is significative to the design and develop of new colchicine site-binding agents.Methods and materials:1. Chemical synthesis:These compounds were synthesized using a one-pot three-component reaction of substituted benzaldehyde, phenol analogs and ethyl cyanoacetate in the presence of piperidine. All of the new compounds described were characterized by1H NMR and mass spectrometry (MS) spectra.(1) Preparation of mHAlA mixture of3-Bromo-4,5-dimethoxybenzaldehyde (0.49g,0.002mol),3-(dimethylamino) phenol (0.27g,0.002mol), ethyl cyanoacetate (0.21ml,0.002mol) and piperidine (0.4mL,0.004mol) was suspended in15mL anhydrous ethanol and stirred at room temperature for4h. After diluting with80mLCH2l2and washing with water, the organic layer was dried over Na2SO4. The Na2SO4was then removed by filtration and the solvent was evaporated. The crude product was purified by column chromatography (hexane/CH2Cl2) to give0.6g mHAl at63%yield.(2) Preparation of mHA6Starting from3-bromo-4,5-dimethoxybenzaldehyde, naphthalen-1-ol, and ethyl cyanoacetate, we then followed the procedure for the synthesis of mHAl, to give0.45g (46%yield) of mHA6.(3) Preparation of mHA11Starting from3-chlorobenzaldehyde,3-(dimethylamino) phenol, and ethyl cyanoacetate, we then followed the procedure for the synthesis of mHAl, to give0.32g (43%yield) of mHA11.2. Cell cultureThe human leukemic HL-60/Bcl-2cell line was obtained from Dr. Kapil N. Bhalla (University of Miami School of Medicine, Miami, FL), which has been stably transfected with pZip-Bcl-2plasmid. Cells were cultured in RPMI1640medium supplemented with10%fetal bovine serum,2mM glutamine,100U/ml penicillin,100μg/mL streptomycin and800μg/mL genticin (G418, Invitrogen, San Diego, CA). Cells were maintained in a humidified5%CO2atmosphere at37℃.3. Mouse bone marrow cells collectionMouse bone marrow samples were obtained from3-month-old C57BL/6female mice (Charles River Labs). Mice were sacrificed with CO2the tibias and femurs were dissected. The tibias and femurs were flushed repeatedly with RPMI1640medium through needle. Mouse bone marrow cells were seperated by Ficoll density gradient centrifugation.4. Isolation and culture of human bone marrow cellsBone marrow specimens were acquired from Diffuse Large B Cell Lymphoma (DLBCL) patients (n=3) via a protocol approved by our Research Ethics Committee for the use of samples for research, and informed consent was obtained from each patient. All of the patients with normal bone marrow were confirmed by pathology. Heparinized bone marrow samples were diluted with RPMI1640medium and overlaid on5ml separation medium and then centrifuged at3000rpm for30min. The bone marrow cells were washed twice and suspended in RPMI1640medium supplemented with10%fetal bovine serum, added in96-well plate (1×105of cells each well).5. Measurement of cell viability via CellTiter-Blue assayHL-60/Bcl-2cells were seeded in96-well plates and treated with various concentrations of these compounds (0.1to3μM). The samples were then incubated at37℃for72h. After incubation, cell viability was measured using a CellTiter-Blue assay kit according to the manufacturer’s instructions.6. Measurement of cell viability via CCK-8assayThe cell viability assays were evaluated by a Cell Counting Kit-8(CCK-8). After overnight culture the bone marrow cells were treated with different concentrations of mHA1, mHA6and mHA11separately, and controls were treated with vehicle (DMSO). After72h treatments, CCK-8solution was added to each well according to the manufacturer’s instructions and optical density (OD) was measured at450nm test wavelength using a microplate reader. All experiments were performed in triplicate.7. Assessment of clonogenicityAfter incubation for24h with various drug concentrations or Vehicle (DMSO) control, cells were collected and diluted with fresh medium, then mixed with2%methylcellulose to make the methylcellulose1.3%and fetal bovine serum30%. A1.5ml volume of this mixture, containing200cells, was seeded into culture dish. Dishes were then incubated in incubator for10-14days. Colonies were enumerated with the aid of an inverted microscope.8. Cell Morphological changeCells were treated with mHAs at1μM for24h and then examined for morphological changes by inverted fluorescence microscopy and photography.9. Molecular modelingThe crystal structure of microtubule in a complex with DAMA-colchicine [PDB reference:1SA0(16)] was used to predict the binding models of microtubule bind with designed compounds. The binding modes of designed ligands1,6,11with microtubule were predicted by using the Autodock4program.10.[3H]Colchicine-tubulin binding assayOne micromolar radiolabeled colchicine [ring C, Methoxy-3H],1%DMSO and various concentrations of test compounds in50μl G-PEM buffer were incubated with1μM tubulin for60min. The binding solutions were filtered through two stacks of DEAE-cellulose filters and washed twice. The radioactivity in the filtrates was determined by liquid scintillation spectrometry. Nonlinear regression was used to analyze the data using GraphPad Prism.11. Tubulin polymerization assayTubulin polymerization assays were conducted using the polymerization assay kit following the manufacturer’s instructions. Briefly,50μl of3mg/ml tubulin (>99%pure) proteins in G-PEM buffer was placed in96-well microtiter plates in the presence of test agents. The absorbance at360/420nm was recorded every60s for1h using a Synergy2microplate reader.12. Immunofluorescence stainingCRL5908cells were treated with1μM mHAl,6, or11for24h. Thereafter, cells were fixed for30min at4℃in4%paraformaldehyde and incubated with0.1%Triton X-100permeabilizing buffer for15minutes. After washing with PBS and blocking with2%BSA in PBS for30minutes, cells were incubated for1h protected from light in1:2000anti-a-tubulin monoclonal antibody and7:1000Rhodamine-Phalloidin-labeled anti-actin antibody in PBS. Cells were then washed with PBS and incubated for30min protected from light with1:200fluorescein isothiocyanate (FITC)-labeled anti-mouse IgG antibody in PBS. Subsequently, all cells were stained with1μg/ml4’,6-diamidino-2-phenyl-indole. Samples were examined under a fluorescence microscope and photographed.13. Cell cycle analysisHL-60/Bcl-2cells (1×106) were treated with1,3, and5μM of mHA1,6,11, respectively, for24h at37℃. Cells were harvested and washed twice with PBS, then resuspended in100μl of PBS and lml of75%cold ethanol and stored at-20℃overnight. After centrifugation, the supernatant was removed. A500μl PI staining buffer containing80μg/mL of propidium iodide,100μg/mL of RNAse A, and1%Triton was added to the samples. The cells were incubated for at least half an hour (avoid light) and then analyzed by flow cytometry with a FACScalibur system using FlowJo7.5analysis software.14. DNA fragmentation analysisHL-60/Bcl-2cells were treated with Vehicle control (DMSO), positive control (colchicine at0.1and1μM), and mHA1,6, or11(at1and5μM), respectively. The plate was incubated for24h, and total DNA was extracted from the cells in each well using an Apoptotic DNA-ladder kit following the manufacture’s instructions. The DNA samples were subjected to2%agarose gel electrophoresis and visualized with ethidium bromide staining. Results:1. Cytotoxicity of new mHA agents toward leukemic HL-60/Bc;-2cellsHuman leukemia HL-60/Bcl-2cells were treated with the series of HA14-1analogs and cell viability assay was performed to get their IC50values. mHAl,6, and11, showed the best anti-tumor activity of the series of compounds and we choose them for further study.2. Structure-activity analysisOn the basis of the biological result, the structure-activity relationship of these compounds was discussed. We digged up that dimethyl amino in C6is similar with benzene and better than hydroxy(-OH); hydroxy(-OH) is better than amino(-NH2); mHA7,15and17are all very poor which indicated that substitution at C7will decrease its activity.3. mHAl,6,11were much more potent than their parent HA14-1compoundHuman leukemia HL-60/Bcl-2cells were treated with diferent concentrations of HA14-1or mHA1,6,11for24h or72h, respectively. Cell viability assay showed that the IC50values of mHAl,6,11were all less than1μM, while IC50of the parent compound, HA14-1, was9.394±0.18μM. Overall, mHA1,6,11were much more potent than their parent HA14-1compound.4. mHAl,6,11were very potent to Human leukemia HL-60/Bcl-2cells while they showed low otoxicity against normal mouse bone marrow cells.Human leukemia HL-60/Bcl-2cells and normal mouse bone marrow cells were treated with diferent concentrations of mHAl,6,11for72h, then cell viability was assessed. The results showed that these compounds had almost no effects on normal mouse bone marrow cells below1μM concentration, while1μM mHAs were able to kill almost half of the malignant HL-60/Bcl-2cells.5. mHAl,6,11showed almost no cytotoxicity on human bone marrow cellshuman bone marrow cells were treated with diferent concentrations of mHAs and DMSO vehicle control for72h, then cell viability was assessed. The results showed that3μM mHAs treatment had almost no effects on normal human bone marrow cells, while3μM mHAs were able to kill almost all of the malignant HL-60/Bcl-2cells.6. The positive control Colchicine showed high toxicity against normal mouse bone marrow cellsHuman leukemia HL-60/Bcl-2cells and normal mouse bone marrow cells were treated with diferent concentrations of colchicine, the colchicine site-binding agent, which was used as the positive control. Cell viability was assessed72h later. The results showed that colchicine was toxic against both malignant and normal cells. Colchicine resulted in the death of30%of normal mouse bone marrow cells at25nM, even though its IC50was only33.5±3.5nM in HL-60/Bcl-2cells.7. Induction of colonogenic cell death by mHA1,6,11.After exposure various concentrations of mHA1,6, or11for24hours, HL-60/Bcl-2cells were then incubated for10-14days, then the number of colonies were measured. The IC50values for the colony inhibition assay were lower than the values for the cytotoxicity assay, which indicated that treatment with mHAs disrupted proliferation and colony forming ability of some cancer cells without causing actual cell death in72hours.8. Specific cell morphological changes in response to mHAl,6,11.mHA1,6and11treatment resulted in specific cell morphological changes. HL-60/Bcl-2cells are suspension cells and typically have a spherical shape. After treatment with mHAs, specific morphological changes including cell elongation, asymmetry, and formation of long pseudopodia were observed. Human lung cancer CRL5908cells changed from spindle shape to multi-angular or nearly-circular shape after treatment with mHAs.9. The docking study showed that mHAl,6, and11could bind at the colchicine site on the microtubule proteinThe crystal structure of microtubule in a complex with DAMA-colchicine was used to predict the binding models of microtubule bind with designed compounds. The results of our docking study showed that mHA1,6, and11could bind at the same site as colchicine on the microtubule protein. mHA1and mHA6adopt a similar binding mode with colchicine, which is slightly different from that adopted by mHA11.10.[3H]Colchicine-tubulin competition binding assay confirmed that mHAl,6,11could compete with colchicine binding to tubulin.[3H]Colchicine-tubulin competition binding assay confirmed that mHAl,6,11could inhibit the combination of [3H]colchicine and tubulin in a dose-dependent manner by competing the colchicine binding site.11. Tubulin polymerization assay showed that mHAl,6,11and colchicine could inhibit the microtubule polymerization while Taxol had opposite effect.Tubulin polymerization assay showed that mHAl,6,11and colchicine could inhibit the microtubule polymerization while Taxol had opposite effect, suggesting that each of the mHAs possessed strong antitubulin polymerization activity.12. Immunofluorescent staining study confirmed that mHAl,6,11could decrease the contents of microtubule and destroy the network of microtubules in cells.Humn lung cancer CRL5908cells were treated with1μM mHA1,6, or11for24hour, then immunofluorescent staining assay were performed. The long microtubule structure along the long axes of the cell in the cytoplasm was disrupted and the microtubule fluorescence intensity was significantly reduced, suggesting the decrease in contents of microtubule.13. G2/M cell cycle arrest caused by mHAl,6,11.Human leukemia HL-60/Bcl-2cells were treated with1μM mHA1,6,11, the distribution of cells in different phases of the cell cycle was determined by flow cytometry24hours later. The results showed that mHAs caused a statistically significant increase in the G2-M cell population.14. DNA fragmentation analysis confirmed that mHAs could induce apoptosisHuman leukemia HL-60/Bcl-2cells were treated with mHAl,6,11and colchicine for24hours, then DNA fragmentation analysis were performed. A clear DNA ladder was observed, indicating that colchicine, and mHAl,6, and11could induce apoptotic cell death in HL-60/Bcl-2cells. Conclusion:1. We developed a series of HA14-1analogs and identified them as a new class of microtubule inhibitors with potent anti-cancer growth activity.2. The structure-activity relationship study of this series of compounds provided references for the design of new microtubule inhibitors in the future.3. These analogs showed a more stable and more potent anticancer growth activity than did authentic HA14-1, with IC50values are all in nM level. And they all showed lower toxicity towards normal tissues which made them possible to be a drug for further study.4. The docking study and [3H]Colchicine-tubulin competition binding assay showed that mHA1,6,11are all binding to the colchicine-binding site on microtubule protein and they are all new microtubule inhibitors.5. Tubulin polymerization assay showed that mHAl,6,11were potent microtubule depolymerizing agent. They worked in a similar manner as colchicine while Taxol was just the opposite.6. Immunofluorescent staining study indicated that mHA1,6,11decreased the microtubule amount and microtubule network structures in the cells.7. Flow cytometry analysis and DNA fragmentation analysis showed that mHAl,6,11induced G2/M cell cycle arrest and lead to cell apoptosis.8. Our study explored the mechanism of this series of HA14-1analogues on malignant cells. They bind at colchicine-binding site on microtubule protein and inhibit microtubule polymerization. The suppression of the microtubule dynamics lead to misdirecting the formation of a functional mitotic spindle, which arrests the cells in G2/M phase, thereby leading to apoptosis of the tumor cell. |
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