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Compound 12 binds to the colchicine-binding site of tubulin and inhibits microtubule polymerisation. (A) Percentage of colchicine-binding inhibition of compound 12 at three different concentrations (1 mM, 5 mM and 10 mM). The tubulin competition-binding assay was performed as indicated in the materials and methods. (B and C) Superimposition of the docked conformation of the colchicine (B) and compound 12 (C) as cyan stick models on top of the X-ray structure of DAMA-colchicine (blue-violet wire model, PDB code: 1SA0). The backbone of tubulin is shown as ribbon representation (a-tubulin, yellow; b-tubulin, green). The key residues interacted with the compound 12 are shown as stick models with the corresponding colour of their ribbon. Hydrogen bonds between the key residues and compound 12 are shown as dash lines.
Source publication
Microtubules are critical elements that are involved in a wide range of cellular processes, and thus, they have become an attractive target for many anticancer drugs. A novel synthesized compound, 12P, was identified as new microtubule inhibitor. This compound inhibits tubulin polymerization through binding to the colchicine-binding site of tubulin...
Contexts in source publication
Context 1
... free base of compound 12P) targets the tubulin-microtubule system and to which site it binds, a colchicine competition-binding assay was performed [23]. The results showed that 12 strongly bound to the [ 3 H]colchicinebinding domain of tubulin, and 40.7%, 62.3% and 84.5% binding inhibition occurred with 12 at 1 mM, 5 mM and 10 mM, respectively ( Fig. 2A). We also performed the molecular modelling studies of colchicine and 12 to investigate the potential pose of compound 12 to the colchicine binding site of a,b-tubulin. The MOE package was employed to investigate the docking between the compound 12 and a,b-tubulin. Before this, the reliability of the docking method was tested. As shown ...
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... Fig. 2A). We also performed the molecular modelling studies of colchicine and 12 to investigate the potential pose of compound 12 to the colchicine binding site of a,b-tubulin. The MOE package was employed to investigate the docking between the compound 12 and a,b-tubulin. Before this, the reliability of the docking method was tested. As shown in Fig. 2B, colchicine could back to colchicine binding site with the similar conformation of the X-ray structure of the colchicine (PDB code: 1SA0) if we re-docking the colchicine. It indicated that our docking method is reliable and it could be employed for the docking of the compound 12. The Docking studies of the compound 12 in Fig. 2C showed ...
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... As shown in Fig. 2B, colchicine could back to colchicine binding site with the similar conformation of the X-ray structure of the colchicine (PDB code: 1SA0) if we re-docking the colchicine. It indicated that our docking method is reliable and it could be employed for the docking of the compound 12. The Docking studies of the compound 12 in Fig. 2C showed that it could occupy the colchicine binding site of tubulin in agreement with the X-ray structure complex of DAMA-colchicine-a,b-tubulin (PDB code: 1SA0, DAMA is N-deacetyl-N-(2-mercaptoacetyl)). Two hydrogen bonds and two s-p conjugate actions were formed between the compound 12 and the residues of tubulin. As shown in Fig. 2C, ...
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... 12 in Fig. 2C showed that it could occupy the colchicine binding site of tubulin in agreement with the X-ray structure complex of DAMA-colchicine-a,b-tubulin (PDB code: 1SA0, DAMA is N-deacetyl-N-(2-mercaptoacetyl)). Two hydrogen bonds and two s-p conjugate actions were formed between the compound 12 and the residues of tubulin. As shown in Fig. 2C, the trimethoxyphenyl moiety of compound 12 was positioned in the binding cavity buried in the b-subunit. The thiol group of Cysb241 formed a hydrogen bond with the oxygen atom of one of the methoxy groups, and two key amino acids of b-tubulin (Leub248 and Leub255) formed hydrophobic interactions (s-p conjugate) with the ...
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... et al. with some modifications [25,26]. In the control samples, which were not treated with any of the tested compounds, the fluorescence intensity at 410 nm increased with time. However, in the presence of 12 at concentrations of 0.312, 0.625, 1.25, 2.5, 5, and 7.5 mM, tubulin polymerisation was inhibited in a concentration-dependent manner (Fig. 2D). The inhibitory concentration that reduces polymerized tubulin by 50% is 1.15 AE 0.21 mM (Fig. 2E), and we also tested the inhibition activity of CA-4 as the reference compound, which IC 50 value was 1.14 AE 0.11 mM. Considering the importance of the tubulinmicrotubule system in the maintenance of cellular morphology, an assay that ...
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... of the tested compounds, the fluorescence intensity at 410 nm increased with time. However, in the presence of 12 at concentrations of 0.312, 0.625, 1.25, 2.5, 5, and 7.5 mM, tubulin polymerisation was inhibited in a concentration-dependent manner (Fig. 2D). The inhibitory concentration that reduces polymerized tubulin by 50% is 1.15 AE 0.21 mM (Fig. 2E), and we also tested the inhibition activity of CA-4 as the reference compound, which IC 50 value was 1.14 AE 0.11 mM. Considering the importance of the tubulinmicrotubule system in the maintenance of cellular morphology, an assay that involved the disruption of microtubule dynamics was performed to reveal whether compound 12P could ...
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... activity of CA-4 as the reference compound, which IC 50 value was 1.14 AE 0.11 mM. Considering the importance of the tubulinmicrotubule system in the maintenance of cellular morphology, an assay that involved the disruption of microtubule dynamics was performed to reveal whether compound 12P could affect microtubule dynamics in living cells. In Fig. 2F, confocal microscopy studies clearly showed the heavy disruption of the microtubule system in A549 cells after treatment with different concentrations of compound 12P for 12 h. The vehicle-treated cells were observed to be predominantly in interphase of the cell cycle, which was characterized by uncondensed chromosomes and the ...
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... all demonstrated that 12P strongly inhibits tubulin polymerisation through binding to the colchicine-binding site of tubulin, disrupts the intracellular microtubule dynamics, interferes with the normal formation of mitotic spindles via the depolymerization of microtubules, which is consistent with the mechanism of most antimitotic agents [48] (Fig. ...
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Citations
... The colchicine competitive binding assay was performed according to our previously reported method (33). In 50 μL of G-PEM buffer, radioactive [ 3 H]-labeled colchicine and 1% dimethyl sulfoxide (DMSO) were added. ...
Background
Microtubules are attractive targets for anticancer drugs. However, the microtubule-targeting agents (MTAs) currently in clinical use exhibit inevitable drug resistance. Therefore, there is an urgent need to discover novel MTAs for the clinical treatment of cancer.
Methods
Bioactive compounds extracted from Lithospermum erythrorhizon were assessed for in vitro anti-proliferative activities against a panel of human cancer cell lines using cell counting kit-8 (CCK-8) assay. Tubulin polymerization inhibition assay, colchicine competitive binding site assay, and immunofluorescence were used to validate the tubulin inhibition effect of acetylshikonin. Flow cytometry, Hoechst staining, and caspase-3 activity evaluation were performed to assess cell cycle arrest and cell apoptosis. 5,5',6,6'-tetrachloro-1,1',3,3'-tetramethylbenzimidazolylcarbocyanine iodide (JC-1) staining and dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining were used to evaluate mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), respectively.
Results
Acetylshikonin exhibited potent anti-proliferative activities against a panel of human cancer cell lines (IC50 values: 1.09–7.26 µM) and displayed comparable cytotoxicity against several drug-resistant cell lines. Further mechanism studies revealed that acetylshikonin induced cell cycle arrest of MHCC-97H cells at G2/M phase, and significantly promoted apoptosis marked by a collapse of MMP and abnormal ROS accumulation.
Conclusions
In this study, acetylshikonin was identified as MTA against hepatocellular carcinoma and can serve as a promising lead compound for further development of anti-cancer drug, underscoring its potential clinical significance.
... In line with our with our findings, it has been reported that CA-4 exhibits an anti-proliferative effect on thyroid cancer cells [24]. After assessing the cytotoxic effects, we proceeded to examine the potential influence of the ROS pathway on cytotoxic capabilities of CA-4, The results of our measurements demonstrated that CA-4 induces intracellular ROS, and scavenging these oxygen species from the cells using NAC can improve the viability of glioma cells [25,26]. Patterson et al. demonstrated that cellular arrest in the G2 phase can lead to an elevation in cellular ROS levels, In this context, our cell cycle analysis revealed G2 arrest in glioma cells following treatment with CA-4, suggesting that this cell cycle arrest could potentially be the source of the elevated concentration of intracellular ROS [27,28]. ...
The most common primary brain malignancy, glioblastoma multiforme, is tremendously resistant to conventional treatments due to its potency for metastasis to surrounding brain tissue. Temozolomide is a chemotherapeutic agent that currently is administrated during the treatment procedure. Studies have attempted to investigate new agents with higher effectiveness and fewer side effects. Combretastatin A-4 (CA-4), a natural compound derived from Combretum caffrum, has been recently considered for its potent antitumor activities in a wide variety of preclinical solid tumor models. Our findings have shown that CA-4 exerts potent anti-proliferative and apoptotic effects on glioma cells, and ROS generation may be involved in these cellular events. CA-4 has imposed G2 arrest in U-87 cells. We also observed that CA-4 significantly reduced the migration and invasion capability of U-87 cells. Furthermore, the gene expression and enzyme activity of MMP-2 and MMP-9 were significantly inhibited in the presence of CA-4. We also observed a considerable decrease in PI3K and Akt protein expression following treatment with CA-4. In conclusion, our findings showed significant apoptogenic and anti-metastatic effects of CA-4 on glioma cells and also suggested that the PI3K/Akt/MMP-2/-9 and also ROS pathway might play roles in these cellular events.
... The effects of 4EN1 compared to 4EN2 were evaluated alongside CA-4 as a representative microtubule depolymeriser (Fig. 4) [78][79][80]. CA-4 has previously been reported as inhibiting tubulin polymerisation by 35 % at 1 μM with activity abolished at 10 μM [81]. The AUC was recorded as an indicator of final polymer mass. ...
... In recent years, our group has been committed to the design and development of indole-containing antitumor agents that act as tubulin polymerization inhibitors [21][22][23][24][25][26]. In this study, as shown in Figure 1, starting from the structure of millepachine, we moved the position of the 2,2-dimethylbenzopyran group and introduced the indole ring to obtain millepachine-indole derivatives. ...
... The biological evaluation applied in this study, including a CCK-8 assay, an in vitro tubulin polymerization inhibition assay, a molecular docking study, colchicine competitive inhibition assay intracellular microtubule morphology detection using immunofluorescence, a cell cycle and apoptosis assay using flow cytometry, a caspase 3 activity evaluation, and intracellular ROS and MMP detection were performed completely under the guidance of our previously published protocols [24][25][26]. These protocols are briefly described in the Supplementary Materials. ...
Millepachine, a bioactive natural product isolated from the seeds of Millettia pachycarpa, is reported to display potential antitumor activity. In this study, novel indole-containing hybrids derived from millepachine were designed, synthesized and evaluated for their antitumor activities. Among all the compounds, compound 14b exhibited the most potent cytotoxic activity against five kinds of human cancer cell lines, with IC50 values ranging from 0.022 to 0.074 μM, making it almost 100 times more active than millepachine. Valuable structure–activity relationships (SARs) were obtained. Furthermore, the mechanism studies showed that compound 14b induced cell-cycle arrest at the G2/M phase by inhibiting tubulin polymerization and further induced cell apoptosis through reactive oxygen species (ROS) accumulation and mitochondrial membrane potential (MMP) collapse. In addition, the low cytotoxicity toward normal human cells and equivalent sensitivity towards drug-resistant cells of compound 14b highlighted its potential for the development of antitumor drugs.
... Seven membered rings as linkers were conducted in the synthesis of novel CA-4 derivatives, and one of these researches innovated a compound St.10 ( Figure 4), which inhibits the tubulin polymerization by binding to the colchicine binding site of tubulin. This compound showed potent antiproliferative activities against various kinds of human cancer cell lines, with IC50 values in nanomolar level, as well as the flow cytometric analysis results showing that this structure can induce cell cycle arrest in G2/M phase and apoptosis in A549 cancer cell line [56]. Another study was conducted on these basic core structures; a series of novel benzoxepins was synthesized and evaluated as anticancer agents, and among this series the most potent compound was St.11 ( Figure 4). ...
Cancer accounts for numerous deaths each year, and it is one of the most common causes of death worldwide, despite many breakthroughs in the discovery of novel anticancer candidates. Each new year the FDA approves the use of new drugs for cancer treatments. In the last years, the biological targets of anticancer agents have started to be clearer and one of these main targets is tubulin protein; this protein plays an essential role in cell division, as well as in intracellular transportation. The inhibition of microtubule formation by targeting tubulin protein induces cell death by apoptosis. In the last years, numerous novel structures were designed and synthesized to target tubulin, and this can be achieved by inhibiting the polymerization or depolymerization of the microtubules. In this review article, recent novel compounds that have antiproliferation activities against a panel of cancer cell lines that target tubulin are explored in detail. This review article emphasizes the recent developments of tubulin inhibitors, with insights into their antiproliferative and anti-tubulin activities. A full literature review shows that tubulin inhibitors are associated with properties in the inhibition of cancer cell line viability, inducing apoptosis, and good binding interaction with the colchicine binding site of tubulin. Furthermore, some drugs, such as cabazitaxel and fosbretabulin, have been approved by FDA in the last three years as tubulin inhibitors. The design and development of efficient tubulin inhibitors is progressively becoming a credible solution in treating many species of cancers.
... We determined that exposure to CuB damaged the microtubule network. In recent years, our group has been committed to the research and development of several anti-tumour drugs derived from natural products that target the tubulin-microtubule system 21,[37][38][39][40][41][42] . We have verified that CuB does not affect purified tubulin polymerization in vitro. ...
Cucurbitacin B (CuB) is a widely available triterpenoid molecule that exhibits various biological activities. Previous studies on the anti-tumour mechanism of CuB have mostly focused on cell apoptosis, and research on the ferroptosis-inducing effect has rarely been reported. Herein, we first discovered the excellent cytotoxicity of CuB towards human nasopharyngeal carcinoma cells and elucidated its potential ferroptosis-inducing mechanisms. Morphology alterations of mitochondrial ultrastructure, as observed via transmission electron microscopy, showed that CuB-treated cells undergo ferroptosis. CuB caused intracellular accumulation of iron ions and depletion of glutathione. Detailed molecular mechanism investigation confirmed that CuB both induced widespread lipid peroxidation and downregulated the expression of GPX4, ultimately initiating a multipronged mechanism of ferroptosis. Furthermore, CuB exhibited anti-tumour effects in vitro by inhibiting cellular microtubule polymerization, arresting cell cycle and suppressing migration and invasion. Finally, CuB significantly inhibited tumour progression without causing obvious side effects in vivo. Altogether, our study highlighted the therapeutic potential of CuB as a ferroptosis-inducing agent for nasopharyngeal cancer, and it provided valuable insights for developing effective anti-tumour agents with novel molecular mechanisms derived from natural products.
... Studies have shown that enhanced ROS level could play a central role in ΔΨm loss [23]. Previous evidences suggest that drugs incubated with cancer cells induce ROS with concomitant loss in ΔΨm and apoptosis induction [24][25][26]. In light of the above reports, it is to suggest that enhanced ROS expression in HT-29 cells upon CBZ treatments could be responsible for the loss of ΔΨm. ...
Background and Aim
Colon cancer ranks fourth and is responsible for causing 10% cancer-related mortality in western countries. Its incidence is rising in many countries due to widespread adoption of the Western diet and lifestyle. Carbamazepine (CBZ) is a FDA-approved antiepileptic drug and a histone deacetylase inhibitor. The aim of this study is to evaluate the cytotoxic potentials of CBZ in human colon cancer cells (HT-29 cells).
Methods
HT-29 cells were treated with 36 and 76 μg/ml of CBZ for 24 h. The cytotoxic effect was evaluated by MTT assay. The intracellular reactive oxygen species (ROS) expression was evaluated through dichloro-dihydro-fluorescein diacetate staining. Morphological changes related to apoptosis were evaluated by dual staining with acridine orange and ethidium bromide. Mitochondrial membrane potential was evaluated by rhodamine 123 staining. Immunofluorescence analysis of caspase 3 was done with confocal microscopy.
Results
CBZ caused significant cytotoxicity in HT-29 cells and the effect was concentration dependent. CBZ treatments also caused significant expression of ROS in HT-29 cells. Dual staining showed early and late apoptotic cells and morphological alterations induced by the CBZ. Confocal microscopic studies confirmed the increased caspase 3 expression in CBZ-treated cells.
Conclusion
CBZ induced apoptosis in HT-29 cell through ROS generation and caspase 3 expression and these results pave the way for further in vivo studies.
... In migrating cells, an asymmetry of the microtubule network is initially established, which generates the feedbacks on Rho proteins to promote the generation of asymmetries in actin contractility and substrate adhesion, resulting in polarization and directional movement of the cell [10][11][12][13]. Microtubules fulfill different roles in cellular processes including intracellular transport, cell division and migration [14][15][16], making them attractive targets for natural toxins in cancer research [17,18]. ...
Background
Cell migration is involved in several pathological processes such as tumor invasion, neoangiogenesis and metastasis. Microtubules are needed in directional migration.
Methods
To investigate the effects of microtubule-binding agents (paclitaxel, vinblastine, colchicine, podophyllotoxin), benzophenanthridine alkaloids (sanguinarine, chelerythrine, chelidonine) and other anti-tumor drugs (homoharringtonine, doxorubicin) on cell migration, we performed the in vitro wound healing assay. The interactions between selected alkaloids and microtubules were studied via U2OS cells expressing microtubule-GFP markers.
Results
The microtubule-binding natural products paclitaxel, vinblastine, colchicine and podophyllotoxin significantly altered microtubule dynamics in living cells and inhibited cell migration at concentrations below apparent cytotoxicity. The benzophenanthridine alkaloid sanguinarine, chelerythrine and chelidonine which affected microtubules in living cells, did not inhibit cell migration. Homoharringtonine (protein biosynthesis inhibitor) and doxorubicin significantly inhibited cell migration, however, they did not exert obvious effects on microtubules.
Conclusion
In this study, we demonstrated that microtubule-binding agents are effective anti-migrating agents; moreover, homoharringtonine and doxorubicin can be referred as anti-migrating agents, but direct microtubule dynamics are not involved in their mode of action. Our study provides evidence that some alkaloids and other microtubule-binding natural products may be interesting candidates for the development of novel agents against metastasis.
Electronic supplementary material
The online version of this article (10.1186/s40360-018-0284-4) contains supplementary material, which is available to authorized users.
... (v/v) matrigel) in order to initiate xenograft animal model [28,29]. Drugs treatments were started when the tumour mass were palpable and the tumour sizes about 100 mm 3 (about 2 week after Hela cells injection). ...
Microtubules are essential for the mitotic division of cells and have become an attractive target for anti-tumour drugs due to the increased incidence of cancer and significant mitosis rate of tumour cells. In this study, a total of six indole 1-position modified 1-indolyl acetate-5-nitroimidazole derivatives were designed, synthesized, and evaluated for their ability to inhibit tubulin polymerization caused by binding to the colchicine-binding site of tubulin. Among them, compound 3 displayed the best ability to inhibit tubulin polymerization; it also exhibited better anti-proliferative activities than colchicine against a panel of human cancer cells (with IC50 values ranging from 15 to 40 nM), especially HeLa cells (with IC50 values of 15 nM), based on the cellular cytotoxicity assay results. Moreover, cellular mechanism studies indicated that compound 3 could induce G2/M phase arrest and apoptosis of HeLa and MCF-7 cells, which were associated with alterations in the expression of cell cycle-checkpoint related proteins (Cyclin B1, Cdc2, and P21) and a reduction in the mitochondrial membrane potential as well as alterations in the levels of apoptosis-related proteins (PARP, Caspase 9, Bcl-2, and Bax) of these cells, respectively. Importantly, in vivo studies further revealed that compound 3 could dramatically suppress HeLa cell xenograft tumour growth compared with vehicle and CA-4 phosphate (CA-4P), and no signs of toxicity were observed in these mice. Collectively, these in vitro and in vivo results indicated that compound 3 might be a promising lead compound for further development as a potential anti-cancer drug.
... JC-1 is mitochondria selective and forms aggregates in normal mitochondria that leads to an orange fluorescence under exciting at 490 nm. However, in depolarized mitochondrial membranes, JC-1 forms monomeric and emits green fluorescence [22]. After EC9706 cells were treated with 2, 4 and 8 μM DS2 for 24 hours, a clear shift in JC-1 staining of the mitochondria from orange to green fluorescence was observed in a dose-dependent manner ( Figure 5A). ...
Ent-kaurane diterpene compounds have attracted considerable attention in recent years due to its antitumor, antibacterial, and antiviral activities. However, the clinical development of natural kaurane diterpenes, for example, oridonin for cancer therapy has been hampered by its relatively moderate potency, limited bioavailability. Herein, we report a newly synthetic analog of natural ent-kaurane diterpene, DS2, which exhibits significantly improved activity of antiproliferation against various cancer cell lines relative to oridonin. DS2 treatment triggers the mitochondria-mediated apoptosis and cell cycle arrest in human esophageal cancer cell lines (EC9706, EC109). Interestingly, normal human esophageal epithelial cells (HEECs) and normal human liver cells (HL-7702) are both significantly more resistant to the growth inhibition by DS2 compared with esophageal cancer cells. The DS2-induced apoptosis in EC9706 cells correlated with the drop of mitochondrial membrane potential (MMP), release of cytochrome c into the cytosol and activation of caspase-9 and -3. The induction of proapoptotic proteins p21 and Bax were also observed in DS2-treated cells. The DS2-induced apoptosis was significantly attenuated by knockdown of Bax proteins. Meanwhile, the DS2 treatment caused generation of reactive oxygen species (ROS) in human esophageal cancer cells, but not in HEECs, which was attenuated by pretreatment with ROS scavenger N-acetylcysteine (NAC). More interestingly, the antioxidants pretreatment completely attenuated DS2 mediated loss of the MMP and apoptosis, as well as Bax expression and growth inhibition. In conclusion, the present study reveals that the mitochondria-mediated cell death by DS2 is associated with Bax regulation and ROS generation, and understanding the function and mechanism of DS2 will help us to design better anti-cancer drugs.
