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. Author manuscript; available in PMC: 2023 May 5.
Published in final edited form as: Eur J Pharmacol. 2022 Mar 23;922:174914. doi: 10.1016/j.ejphar.2022.174914

Characterization of Caerulomycin A as a Dual-targeting Anticancer Agent

Lingying Tong 1,2, Weichao Sun 1, Shiyong Wu 1,2,3, Yong Han 1
PMCID: PMC9077901  NIHMSID: NIHMS1792962  PMID: 35337812

Abstract

Caerulomycin A (CaeA), isolated from actinomycetes, has a featured 2,2′-bipyridine core structure. Based on the results of in silico drug-protein docking analysis, CaeA shows potential ligands for interacting with both tubulin and DNA topoisomerase I (Topo-1). The result was confirmed by cell-free tubulin polymerization assay and Topo-1 activity assay. In vitro assays also demonstrated that CaeA increases the polymerization of tubulin and increases cell size. In addition, CaeA inhibits cell viability and growth of various cancer cells, yet exhibits low cytotoxicity. CaeA also affects paclitaxel-resistant cancer cells and synergizes the effect with paclitaxel in reducing cancer cell colony formation rate. In vivo experiments confirm the effect of CaeA on reducing tumor size and weight in nude mouse inoculated with tumor cells with no noticeable side effects. Taken together, our data demonstrate that CaeA is a potential potent agent for cancer treatment through tubulin and Topo-1 dual-targeting with little side effects.

Keywords: Caerulomycin A, Tubulin, Topoisomerase I, Dual-targeting Anticancer Agent

Graphical Abstract

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Introduction

Microorganisms are rich natural resources of identifying anticancer drug candidates [1, 2]. In the past 20 years, thousands of microbial metabolites have been found to inhibit tumor growth or have cytotoxic effect on tumor cells based on cellular and animal studies [3, 4]. Based on their structures, these compounds are divided into enediynes [5], glycopeptides [6], anthracyclines [7], macrolides [8], benzodipyrroles [9], quinoxalines [10], etc. More than 10 of them have significant curative effects and have been developed to chemotherapeutic drugs [3]. However, most of these anticancer compounds have no specificity on targeting proliferating cells, thus have a non-specific cytotoxic effect on both fast-proliferating cancerous cells and slow- or non-proliferating normal cells [11]. The lack of specificity to cancer cells leads to relatively high toxic side effects. In here, we reported that Caerulomycin A (CaeA), isolated from marine-derived Actinoalloteichus cyanogriseus DSM 43889, features a unique 2,2′-bipyridine core structure, which is different from any previous identified anti-tumor drugs [12]. The 2,2′-bipyridine core structure shares structural similarity to biphenyl compounds, which has the potential to bind to tubulins and DNA topoisomerase I (Topo-1).

Tubulins are the construction units of microtubules, which play important roles in many cellular processes. They maintain the structure of cells and form cytoskeleton with microfilaments and intermediate fibers [13]. They are also involved in cell division by involving in spindle formation [14]. Thus, microtubule is an important target for antitumor drugs [15]. The drugs targeting on microtubules are mainly divided into two categories: one group of compounds promote tubulin polymerization and stabilize formed microtubules (inhibition of microtubule depolymerization). Compounds, originally isolated from natural sources, stabilize microtubules including paclitaxel and its analogues [16], epothilones [17], taccalonolides [18] and discodermolides [19]. The other group of natural compounds, such as colchicines [20], vinca alkaloids [21], dolastatins [22], hemiasterlin [23] and combretastatins [24], inhibit tubulin polymerization [25]. DNA Topoisomerases are essential enzymes for DNA replication. Considering the rapid cell division and high DNA replication rate in cancer cells, blocking DNA topoisomerases is generally considered to be a potent method to inhibit cancer growth [26]. Camptothecin (CPT) and doxorubicin are the two most popular anti-cancer agents targeting topoisomerase I and II respectively [27, 28]. Both compounds are marketed and widely used in chemotherapy.

In this report, based on in silico drug-protein docking analysis, we provided in vitro evidence that CaeA is a potent anticancer compound that promotes tubulin polymerization and inhibits Topo-1 activity. We also showed that CaeA affects paclitaxel-resistant cancer cells and may have a synergetic effect when used together with paclitaxel. Our in vivo data, agreed with the cell-based data, showed that CaeA significantly reduced the tumor progression in nude mice inoculated with human tumor cells. Based on our data, we believe that CaeA shows its potential as an anti-cancer agent by its dual targeting tubulin and Topo-1.

2. Materials and methods

2.1. Fermentation and Isolation of Caerulomycin A

Actinoalloteichus cyanogriseus DSM 43889 was used as the Caerulomycin A produce strain. Trypticase soy broth agar was used as seeds culture medium. The fermentation was performed on ISP3 (10 L) agar plates for 11 days at 28°C. The medium was diced and extracted with AcOEt/MeOH (80 : 20). The organic layer was collected by filtration and yielded crude extract upon evaporation. The crude extract was partitioned between water and EtOAc (1: 1) until the EtOAc layer was colorless. The EtOAc extract was then partitioned between MeOH and petroleum ether. The MeOH layer was concentrated in vacuum to afford a crude brown syrupy extract (16.8g). The extract was separated by MPLC (30g RP-18 silica gel; 30% - 100% MeOH, 1 L for each gradient) to afford 5 subfraction (Fr. 1 - Fr. 5). Fr. 3 was subject to column chromatography (CC) on Sephadex LH-20 (in MeOH) to afford 8 subfraction (Fr. 3a - Fr. 3h). Fraction 3e was subject to purification on HPLC (C-18 column, 9.4×250mm, 5 μm; 37% acetonitrile) to afford Caerulomycin A (57 mg, purity 99.3%).

2.2. Computer modeling

The three-dimensional (3D) structure of the Tubulin-Colchicine complex (4O2B) and Human Topo-1 (PDB ID: 1T8I) was downloaded from the RCSB Protein Data Bank (www.rcsb.org) [29, 30]. Molecular docking was performed to investigate the binding mode between CaeA and receptor using Autodock vina 1.1.2 [31]. The 2D structure of CaeA was drawn by ChemBioDraw and converted to 3D structure by ChemBio3D software. The AutoDockTools 1.5.6 package was employed to generate the docking input files [32, 33]. The search grid of the Tubulin-Colchicine complex site was identified as center_x: 13.722, center_y: −16.528, and center_z: −40.611 with dimensions size_x: 20, size_y: 22, and size_z: 30. The search grid of the Topo-1 site was identified as center_x: 21.289, center_y: −2.905, and center_z: 27.814 with dimensions size_x: 32, size_y: 38, and size_z: 22. In order to increase docking accuracy, the exhaustiveness value was set to 20. Default parameters were used for Autodock vina. The best-scoring pose as judged by the Vina docking score was chosen and visually analyzed using PyMoL 2.4.0 Open-Source software [34].

2.3. Cell culture and compound preparation

All the human tumor cells, A375, A549, H1299, HepG2, HT29, HL-60 and M624 (ATCC) were grown in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% Penicillin-Streptomycin (Hyclone) and incubated at 37 °C with 5% CO2. CaeA was prepared in DMSO in 1000X stock and dissolved in complete medium at indicated concentration. Cells were incubated in medium with CaeA for indicated time before analysis.

Paclitaxel-resistant A375 cell was established by incubating A375 cell in paclitaxel-containing medium from 2 nM to 50 nM. Cells were incubated in 2 nM paclitaxel for one week and paclitaxel concentration was doubled every week afterwards. The final concentration of paclitaxel was 50 nM and cells were incubated in this concentration for 2 weeks before experiments.

2.4. Tubulin Polymerization Assay

Tubulin polymerization assay biochem kit (Cytoskeleton, Inc) was used to study the effect of CaeA on tubulin polymerization. Manufacturer protocol was followed. Briefly, HTS porcine tubulin protein (>97% pure) was dissolved in general tubulin buffer at the concentration of 5 mg/mL, with 1 mM GTP in the solution. 10 μL general tubulin buffer and 10 μL tubulin glycerol buffer were added into each well followed by 10 μL DMSO-dissolved paclitaxel (final concentration 10 μM), nocodazole (final concentration 10 μM) or CaeA (indicated concentration). The plate was then incubated for 2 min at 37 °C. 100 μL of tubulin solution was then added into each well and the plate was immediately read in the spectrophotometer at absorbance 340 nm with 1 min reading interval for 60 min at 37 °C.

2.5. Colchicine Displacement

The ability of CaeA to displace colchicine from tubulin was tested by fluorescent and LC-MS together. Briefly, for the fluorescent method, 2 mM tubulin with 2 mM colchicine were incubated for 2 hours at 37°C containing with vehicle (DMSO) or negative control (100 µM Vinblastine) or CaeA (20 mM)[35]. The fluorescence intensity was read at 380ex / 438em using Cytation 3 Cell Imaging Reader (Bio-tek). The fluorescence of tubulin and colchicine as control and the fluorescence values were normalized by subtracting the 2 mM tubulin without colchicine. For the LC-MS method, after 2 mM tubulin with 2 mM colchicine were incubated for 2 hours at 37°C, ultrafiltration to remove the free colchicine, repeated until colchicine could not be detected in the filtrate (Fig. 2C–a). Then CaeA was added until the final concentration to 20 mM, incubated for another 2 hours at 37°C, ultrafiltration and filtrate were analyzed by HPLC-MS. The HPLC-MS analysis was performed on an Agilent 1260–6120 LC/MS system with a Kinetex C18 (100 mm × 2.10mm × 2.6nm) column, the mobile phase was 25% Acetonitrile (containing 0.1% formic acid), the MS was in positive mode and choose m/z = 400.4 as selected ion (SIM).

Figure 2. CaeA affects tubulin polymerization and Colchicine Displacement.

Figure 2.

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(A): Purified tubulin was dissolved in tubulin general buffer and glycerol buffer using HTS-tubulin polymerization assay kit. 10 uM and 200 uM CaeA were added into the reaction buffer and the effect in manipulating tubulin polymerization was determined by the recording the absorbance at 340 nm in 1 min interval for 60 min. Paclitaxel and nocodazole were used as microtubule stabilizer and de-stabilizer controls respectively. The effects of CaeA on colchicine binding were evaluated by (B) fluorometric and (C) HPLC-MS method.

2.6. Topoisomerase activity assay

The topoisomerase activity is determined by Topoisomerase I assay kit (TopoGEN, Inc) following instructions of manufacturer. Briefly, 50 ng plasmid DNA was incubated with 1 Unit topoisomerase with or without compounds in the reaction solution and incubated at 37 °C for 30 min. Stopping buffer was then added into the reaction solution and DNA was separated on agarose gel without ethidium bromide. Gels were stained in ethidium bromide solution, washed and photographed. Camptothecin (CPT), a known Topo-1 inhibitor, was used at 50 μg/mL as a positive control.

2.7. Immunofluorescent staining of a-tubulin

A375 cells were seeded on cover slip and treated with CaeA for 24 hours. After treatment, cells were fixed with 10% formalin for 10 min at room temperature, rinsed with PBS three times and permeabilized with 0.1% Triton X-100 in PBS for 5 min on cover slip. Blocking buffer (2 mg/mL BSA in PBS) were added to cell for 1 h before incubating with mouse anti-a-tubulin antibody (Cell Signaling) for overnight at 4 °C. After three times washing with PBS, cells were incubated with a fluorescein-conjugated horse anti-mouse antibody (Vector Labs) for 1 h, washed with PBS and mounted with ProLong Gold Antifade Reagent with DAPI (Invitrogen). The pictures were taken by Zeiss upright confocal microscope and analyzed with Image J (NIH).

2.8. Cell viability assay

A375 cells (5 × 103) were seeded into 96-well plate and incubated overnight for attachment. Cell medium was then replaced by fresh medium containing CaeA or DMSO. At indicated time point (24, 48, or 72 hours) after treatment, medium was removed and 100 μL resazurin solution (final volume 0.04%) was added into the well. After 45 min incubation at 37 °C, fluorescence intensity was read at 560ex / 590em using Cytation 3 Cell Imaging Reader (Bio-tek).

2.9. Clonogenic assay

A375 cells were seeded into plates at the density of 800 cell per 60 mm dish and incubated for attachment overnight. The medium was then replaced with medium containing CaeA or/and paclitaxel at indicated concentration and continued incubating for 7 days in incubator. Cells were then fixed with cold methanol for 10 min at −20 °C and stained with 1% crystal violet in 25% methanol for 10 min at room temperature. Fixed cells were then rinsed with water and air-dried at room temperature. Colonies with a size greater than 0.4 mm were counted by Kodak IS in vivo F system equipped with Kodak Molecular Imaging Software (Eastman Kodak).

2.10. Cell cycle analysis

A375 cells were seeded in 60-mm tissue culture dish and starved in FBS-free medium 24 hours to synchronize cell cycle. The FBS-free medium was then replaced with regular medium containing DMSO or indicated concentration of CaeA. After 24 hours of incubation, cells were harvested in pellet by trypsin and washed twice with ice-cold PBS. Cells were then fixed in 70% ice-cold ethanol for at least 30 min at 4 °C, followed by 2 times wash in PBS. 50 μL RNase (100 μg/mL) and 200 μL PI (50 μg/mL) were added to degrade RNA and stain DNA. Stained cells were analyzed using Accuri C6 flow cytometer (BD Biosciences).

2.11. Western blot

Cells were lysed with Nonidet P-40 (NP-40) lysis buffer (2% NP-40, 80 mM NaCl, 100 mM Tris-HCl pH 8.0, 0.1% SDS) with proteinase inhibitor mixture (Complete™, Roche Molecular Biochemicals) at indicated time point after treating with CaeA. Cell lysate was incubated on ice for 15 min and then centrifuged at 16,000 g at 4 °C for 15 min. Protein concentration was measured by Protein DC Assay kit (Bio-Rad Laboratories). Equal amounts of protein were subjected on SDS-PAGE and transferred to nitrocellulose membrane. The membrane was then blocked in 5% milk in Tris buffered saline plus Tween 20 (TBST) for 45 min and probed with anti-phospho-histone 3 (Cell Signaling), or anti-b-actin (Santa Cruz) at 4 °C overnight. After washing with TBST, the membrane was incubated with corresponding HRP-conjugated anti-rabbit or anti-mouse antibody for 45 min at room temperature. Membrane was then washed three times in TBST followed by two times wash in TBS, and developed in West Pico Supersignal chemiluminescent substrate (Pierce). The images were captured and analyzed by Odyssey Imager and Odyssey image studio software (LI-COR).

2.12. IC50 analysis.

IC50 was determined using MTT based toxicology assay kit (Sigma). Briefly, cells were plated into 96 well plate and incubated overnight. The cells were then treated with different concentrations of CaeA. After 24 hours, MTT solution (1:10 dilution) was added and incubated with the cells for 3 h before measuring absorbance at 570 nm.

CaeA was also evaluated at five concentrations on the NCI-60 cell line panel by the National Cancer Institute Developmental Therapeutics Program (NCI/DTP).

2.13. In vivo xenograft model and treatment

Female homozygous nude mice (Nu/J Foxn1 nu) at the age of 5 weeks were purchased from the Jackson Laboratory and quarantined for one week in Ohio University animal facility before tumor inoculation. A375 cells were prepared in PBS and mixed with Matrigel (Corning) at equal volume, 100 μL of the mixture (5x105 cells) was injected into mouse subcutaneously. After injection, mice were randomly divided into control and treatment groups. Control vehicle or CaeA (5 mg/kg) was administrated via intraperitoneal injection three times a week starting from the second day of tumor injection. When tumors became palpable, the concentration of CaeA was increased to 10 mg/kg. Approximately 4 weeks after tumor injection, mice were euthanized, and tumors were surgically removed for comparison. All the experimental protocols with animals were approved by the Institutional Animal Care & Use Committee of Ohio University (Protocol Number: 19-H-015).

2.14. Statistics

The data were expressed as the mean±SD with each experiment repeated at least three times. The statistical significance of differences for the mean values between groups was determined by Student’s t-test. Differences with a p-value of less than 0.05 were considered statistically significant.

3. Results

3.1. Computation modeling of CaeA binding with tubulin and topoisomerase I.

Because of tubulin presents a high number of binding sites[36], so we considered several potential molecular binding pockets, and some possibilities were ruled out by molecular docking. CaeA features the core structure of 2,2’-bypyridine (Fig. 1), which is similar to the structure of biphenyl. Since biphenyl compounds have been reported to interact with tubulin [37] [38], we suspect that CaeA may also interact with tubulin. To prove this hypothesis, a docking study of CaeA was performed using Autodock Vina1.1.2. The results revealed that CaeA adopts a compact conformation to bind inside of the colchicine-binding pocket at the intra-dimer interface between the α- and β-tubulin subunits (4O2B, Fig. 1B). The computational modeling suggests that the methoxy group of CaeA forms a hydrogen bond with αVal181, the oximido group contacts the amino acid residues (αAsn101) and the pyridine ring contacts three hydrophobic amino acid residues (βLeu255, βCys241 and βLeu248) via hydrophobic interactions (Fig. 1B).

Figure 1. CaeA in silico interaction with tubulin and Topo-1.

Figure 1.

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(A): Chemical structure of CaeA; (B): CaeA was docked into tubulin binding pocket; (C): CaeA was docked into Topo-1 binding pocket.

In addition, CaeA also shares some structural similarity to CPT, a known Topo-1 inhibitor, thus we also performed a docking study between CaeA and Topo-1 using Autodock Vina1.1.2. The results indicated that CaeA uses a compact conformation to bind inside the CPT binding pocket (1T8I, Fig. 1C). It also suggests that the methoxy group of CaeA forms a hydrogen bond with Thr718, the oximido group contacts the DNA bases (DT10 and TGP11) with two hydrogen bonds and the pyridine ring contacts the DNA bases TGP11 via two hydrogen bonds (Fig. 1C).

3.2. CaeA interacts with tubulin as a positive effector on tubulin polymerization

Since in silico assay indicates the potential binding between CaeA and tubulin, we performed cell-free tubulin polymerization assay (Cytoskeleton, Inc) to confirm the result of computational modeling, using paclitaxel (10 mM) and nocodazole (10 mM) as controls of microtubule stabilizer and de-stabilizer respectively. The data showed that CaeA (10 to 200 mM) increased the amount of tubulin polymer by about 20% at 30 min and about 10% at 60 min post-treatment, while paclitaxel (10 mM) increases the amount of tubulin polymer by 64% and 28% at 30 and 60 min respectively (Fig. 2; Table S1). Furthermore, the sigmoid growth plot of tubulin was lost in the presence of paclitaxel, but enhanced in the presence of CaeA (Fig. 2A). These results indicate that CaeA acts more like a positive effector than a stabilizer as paclitaxel does on the tubulin polymerization. In the next colchicine displacement experiment, it was found that CaeA reduced the fluorescence intensity of the colchicine/tubulin complex at a concentration of 20 mM (Fig. 2B), and HPLC-MS experiments also showed that CaeA could replace colchicine that had been bind to tubulin (Fig. 2C), confirming that CaeA occupancy the binding site of colchicine.

3.3. CaeA changes size and morphology of melanoma cells

As the cell-free assay data indicated CaeA affected tubulin polymerization, we further studied the effect of CaeA on tubulin density in cells using immunofluorescent staining method. Our data showed that CaeA decreased the tubulin density by 20% compared to control group while paclitaxel increased the tubulin density by 40%. The combined treatment of CaeA and paclitaxel did not show a statistically significant change compared to control group (Fig. 3A). However, the CaeA treatment increased the size of A375 human melanoma cells (Fig. 3). We confirmed that the size of the cells is approximately doubled after the treatment of CaeA using flow cytometry as both forward scatter (FSC) and side scatter (SSC) increase (Fig. 3B, DSMO as blank). As microtubule dynamics is one of the critical factors in determining cell size and morphology [39], our results suggest that the microtubule dynamics altered by CaeA leads to a change of cell size and morphology of the cells without increasing the microtubule density in the cells.

Figure 3. CaeA affects cell morphology and tubulin density.

Figure 3.

Figure 3.

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A375 cells were treated with 200 nM CaeA for 24 hours. (A) Up: A375 cells were treated with 200 nM CaeA for 24 hours and cells were fixed and stained with a-tubulin and DAPI. Images were taken under Zeiss confocal microscope. Bottom: Quantification of relative a-tubulin fluorescent intensity in confocal pictures using ImageJ. *p<0.05 compared to control. (B): Cell morphology (FSC and SSC) determined by flow cytometry.

3.4. CaeA inhibits the activity of Topo-1

To confirm the modeling of CaeA- Topo-1 binding, we scrutinized the interaction between CaeA and Topo-1 using enzyme assay with CPT as a positive control. The results showed that by adding Topo-1 alone, it cut about 85% of the plasmid DNA into its relaxed form and CPT reduced the cleavage rate to approximate 25%. The effect of CaeA on Topo-1 was tested in a dose dependent manner from 0.05 μM to 5 μM. CaeA slight inhibited the activity of Topo-1 at 0.05 μM, but reduced the Topo-1 activity to about 50% and 40% at concentration of 0.5 μM and 5 μM. The data demonstrated that CaeA inhibits Topo-1 activity in dose dependent manner from 0.05 μM to 5 μM (Fig. 4).

Figure 4. CaeA inhibits the activity of Topo-1.

Figure 4.

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Plasmid DNA was incubated with Topo-1 and various concentration with CaeA (from 0.05 μM to 5 μM) for 30 min at 37 °C. (A): Gel photo of the DNA after treatment. (B): Quantitative analysis on the intensity of the supercoil DNA band. *p<0.05 compared to Topo-1 treating alone.

3.5. CaeA induces death and inhibits colony formation of melanoma cells.

Both the process of tubulin polymerization and the activity of Topo-1 are involved in cell proliferation. Considering the rapid growth of cancer cell, CaeA could be a potential anti-cancer agent by targeting both tubulin and Topo-1. Therefore, we investigated the role of CaeA in cancer cells started from melanoma cell lines as previous study showed that alteration of microtubule dynamics could inhibit melanoma progression [40]. First, cell viability assays were performed using A375 human melanoma cells. Our data show that the cell viability reduces from 92% to 64% in 24 h, 80% to 49% in 48 h; and 73% to 48% in 72 h with 50 to 500 nM CaeA (Fig. 5A, Tabel S2). In addition, we performed clonogenic assay and our data demonstrated that as low as 50 nM CaeA could decrease the colony formation number by 85% and 100 nM CaeA almost completely inhibits colony formation in A375 melanoma cells (Fig. 5B). These results indicated that CaeA is a relatively potent compound to reduce cancer cell viability.

Figure 5. CaeA reduces cancer cell viability in both time and dose-dependent manner and reduces cancer cell colony formation and sensitizes cell responses to paclitaxel.

Figure 5.

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(A) CaeA with indicated concentration was added into A375 human melanoma cells and the cell viability was determined by resazurin assay at indicated time point after CaeA treatment. The error bar represents three to five sets of independent experiments. *p<0.05 compared to control. (B) Up: Representative pictures of clonogenic assay. A375 cells were incubated with indicated concentration of CaeA and pictures were taken after colony staining. Bottom: Quantitative analysis of the colonies formed in the dish incubating with indicated concentration of CaeA. The error bar represents three sets of independent experiments. *p<0.05 compared to control.

3.6. CaeA arrests cell cycle at G2/M phase.

As both tubulin and Topo-1 regulate cell cycle, we analyzed the effect of CaeA on cell cycle phase distribution on cancer cells [41] [42]. Cell cycles were synchronized in FBS-free medium for 24 h before being treated with CaeA in a dose-dependent manner. Our data showed the population of the cells in G2/M phase increased from 31% (control) or 28% (DMSO) to 40–69.9% (100–500 nM CaeA) at 24 h post-treatment (Fig. 6A). Meanwhile, the population of the cells in G1 phase was statistically significantly reduced from 54% (control) or 56% (DMSO) to 42–11% (100–500 nM CaeA) at 24 h post-treatment (Fig. 6A). At 500 nM CaeA treatment, the S phase induced to 20% from 15% of control cells. To further confirm the cell cycle arrest data, we tested the expression level of serine 10-phosphorylated human histone H3 (p-histone H3), a mitosis marker [43]. Our data showed that the level of phospho-histone H3 decreased 10%, 46% and 82% after treating with 100, 200 and 500 nM CaeA respectively (Fig. 6B). These results indicated that CaeA inhibits melanoma cell mitosis through the induction of cell cycle arrest mainly at G2/M phase, which agrees with other tubulin polymerization interrupting compounds.

Figure 6. CaeA arrests cell cycle at G2 phase.

Figure 6.

Figure 6.

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(A): Cell cycle analysis of A375 cells treated with CaeA in a dose-dependent manner using Accuri C6 flow cytometer. (B) Up: Western blot analysis of the phospho-Histone H3 protein level in cells after CaeA treatment. Bottom: Quantitative analysis of the band density of phosphor-histone 3 normalized to beta actin. *p<0.05 compared to control.

3.7. CaeA inhibits cancer growth in Paclitaxel -resistant cell line

Paclitaxel is a widely used chemo-agents used to treat a number of types of cancer. Yet, as with many other chemotherapeutic agents, resistance remains as one major concern of paclitaxel. Though both CaeA and paclitaxel targets on microtubules, CaeA affects microtubules in a different mechanism than paclitaxel. In addition, CaeA also targets on Topo-1. Therefore, we tested the efficacy of CaeA in paclitaxel-resistant cancer cells. Our data showed that A375 cells responded to paclitaxel, yet A375 paclitaxel-resistant (A375 taxol-res) cells did not. When treating both cell lines with CaeA, the viability decreased to about 50% in both cell lines with no statistical difference. This data indicated that paclitaxel-resistant cancer cells responded to CaeA in similar way to non-resistant cells (Fig. 7A).

Figure 7. CaeA affects paclitaxel-resistant cell.

Figure 7.

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(A): A375 and A375 paclitaxel-resistant (A375 Taxol-res) cells were treated with indicated concentration of paclitaxel and CaeA for 24 hours. Cell viability was determined by resazurin assay. The error bar represents three to five sets of independent experiments. *p<0.05 compared to A375 cells. (B): Quantitative analysis of the clonogenic assay of A375 cells treated with indicated concentration of CaeA with or without paclitaxel. All colony numbers were normalized to control group. The error bar represents three sets of independent experiments. *p<0.05 compared to no CaeA treatment group.

In addition, we also performed clonogenic assay using combined treatment of both CaeA and paclitaxel. The data showed that when combining the treatment of CaeA and 1 nM paclitaxel, the colony formation rate decreased from 88% (1nM paclitaxel) to about 60%. Treating the cells with 2 nM paclitaxel alone further reduced the colony formation rate to 68%, while combining with CaeA, the rate further reduced to about 40% (Fig. 7B). These results suggest that CaeA may have a synergetic effect when used together with paclitaxel.

3.8. CaeA inhibits growth of various cancer cells.

To determine the potential of CaeA as a broad anti-cancer drug, we determined the IC50 of CaeA against various cancer cells. Our data showed that CaeA inhibited the growth of all tested cancer cell lines, including lung cancer (A549, H1299), liver cancer (HepG2), colon cancer (HT29), lymphoblast (HL-60), and melanoma (M624), at a concentration between 0.85–5.60 mM (Table S3). In addition, CaeA was also evaluated by NCI-60 cell line panels. The results show that CaeA inhibits the growth rate by 50% (GI50) of most cell lines at about 300 nM (Supplementary Fig. S7).

3.9. CaeA inhibits tumor growth in animal model.

To evaluate the effect of CaeA in tumor growth inhibition, A375 melanoma cells were injected in nude mice. Tumor size and tumor weight were compared between control group and CaeA treated group after mouse euthanizing and tumor removal. The CaeA treated group showed a significant reduction in both tumor size, the average tumor weight was reduced from 0.75 g to 0.29 g by treating with CaeA (Fig. 8, Tabel S4).

Figure 8. CaeA inhibits tumor growth in vivo.

Figure 8.

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A375 melanoma cells (5×105 cells) were inoculated on nude mice. (A): Comparison of tumors surgically removed from mice. (B): Tumor weights measured after tumor removal. *p<0.05 compared to control.

4. Discussion

In recent decades, the discovery of anti-tumor drugs has been focused on finding or designing highly selective compounds acting on single target [44]. However, in treating cancers, the outcome of drugs with single target are often unsatisfactory and prone to induce drug resistance [45]. Furthermore, the proportion of new drug candidates being successfully marketed after clinical trials declines [46]. In recent years, multi-target drug therapy emerges owing to the development of systems biology [47]. This approach can overcome the limitations of single-target drugs, regulate multiple signaling pathways simultaneously, and relatively invulnerable to induce drug resistance. Being noticed by its advantages, multi-target drugs has been applied in the treatment of many major diseases including cancer therapy [48].

Microtubules and DNA topoisomerases are two ubiquitous components in cells involving in cell proliferation. Both serve as targets for anti-cancer agents because of the higher proliferation rate of cancer cells compared to normal ones. Microtubules, due to its extensive biological functions and unique kinetic properties, play a critical role in maintaining regular cell function and serve as target for antitumor agents development [15]. Many compounds have been isolated and characterized as tubulin-targeting anticancer drugs from natural resources. Most types of these compounds are from plants [16, 18, 19, 21–25] besides one type from microorganism [17]. DNA topoisomerases are enzymes regulating DNA replication and transcription, which are also critical in rapid proliferating cancer cells. Natural compound derived compounds, such as CPT and anthracycline, are marketed and widely used in treating various cancer types [49, 50].

In this report, we provided evidence that CaeA, a compound isolated from microorganism, has a unique molecular structure different from any known tubulin- or topoisomerase-binding molecules, and it could be a potential anticancer drug by dual-targeting tubulin and Topo-1. Because of its structural similarity to known tubulin- and topoisomerase-binding compounds, in silico analysis was performed and revealed that CaeA has high affinity with the colchicine-binding pocket on tubulin (Fig. 1B) and CPT binding pocket on Topo-1 (Fig. 1C). The computational modeling results were confirmed by cell-free and cell-based assays to further prove the binding between CaeA- tubulin and CaeA- Topo-1 (Figs 2–4).

CaeA shows potent antitumor activities in cancer cell lines, with IC50 of < 6 mM in against various cancer cells (Table S3). These data are comparable to the potencies of current tubulin-targeting anti-cancer drugs on the market, which have a range of IC50 from 10 nM to 5 µM [51–54]. The NCI-60 screening results showed that the GI50 of CaeA is relatively low (~10−7 M) while the LC50 of CaeA is relatively high (>10−4 M). This suggest that CaeA has low cell cytotoxicity but effectively inhibits cell growth. This also agrees with our data that we didn’t notice significant cell apoptosis or necrosis at our tested concentration within 72 hours in cell lines listed in Table S3, though we cannot rule out the possibility that at higher compound concentration, apoptosis or necrosis may occur.

In addition, the results also agree with our data that CaeA interacts with tubulin and Topo-1, both are more involved in rapid proliferating cells but less active in slow proliferating cells. In addition, we also showed that CaeA promotes cell cycle arrest, reduces cell viability, and inhibits colony formation, at nanomolar level (Figs. 4–7). The major cell cycle arrest at G2/M phase may be caused by interruption of tubulin polymerization. It is worth to notice that the increase in S phase at high concentration of CaeA (500 nM) may be caused by the inhibition of Topo-1, as previous report indicated that Topo-1 inhibition leads to both S and G2/M phase arrest [42, 55]. In addition, we noticed that the cell size increases with the treatment of CaeA, which could be a result of interrupting both Topo-1 and tubulin. The inhibition of topoisomerase cause polyploidy in cells; in the meanwhile the interruption of microtubules causes the incompetence in cell mitosis, both factors may contribute to cell size increasing [56] [57].

Drug resistance is one of the biggest challenges in cancer therapy. Paclitaxel (Taxol), a widely used medication for various cancers, also encounters this problem with uncertain reason though a couple of mechanisms have been proposed [58, 59]. Though both compounds targeted on microtubule, CaeA also targets Topo-1 and further reduced the cell viability in paclitaxel-resistant cells. When treating together with paclitaxel, CaeA shows a synergetic effect in reducing cell viability, suggesting it could potentially be used to increase the potency of paclitaxel and to treat paclitaxel resistant cancers (Fig. 7).

Overall, this study demonstrated that CaeA could be a new type of therapeutics for treating various types of cancers with a dual mechanism of promoting tubulin polymerization and inhibiting topoisomerase. It also has the potential to be used for taxol-resistant cancer treatment. Further characterization of CaeA-related compounds, such as the structural modification and optimization, could provide novel perspective for the development of a new class of compounds with the 2,2′-bipyridine core structures for cancer treatment.

Supplementary Material

1

Highlights.

Caerulomycin A is a multitarget antitumor drug that inhibits both tubulin and topoisomerase I.

Caerulomycin A shows a broad inhibition on cell viability and growth of various cancer cells yet exhibits low cytotoxicity.

Caerulomycin A reduce the tumor size and weight in nude mouse inoculated with tumor cells with no noticeable side effects.

Acknowledgements

We acknowledge the use of the Ohio University Heritage College Microscopy Core for confocal images and the help from Dr. Zhenyu Li (Shandong University) for the use of AutoDock Vina. This work was partially supported by American Cancer Society Postdoctoral Fellowship PF-1605101-NEC (to L.T.); NIH R01 1R01ES030425–01A1 (to S. W. and L.T.); and research start-up fund from Edison Biotechnology Institute at Ohio University (to Y. H.).

Footnotes

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CRediT author statement

Lingying Tong: Methodology, Software, Data curation, Writing- Original draft preparation. Weichao Sun: compound preparation. Shiyong Wu: Writing- Reviewing, Validation. Yong Han: Conceptualization, Supervision, Writing- Reviewing and Editing.

Conflicts of interest: The authors declare no potential conflicts of interest.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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