br All animal studies were approved
All animal studies were approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University. Male BALB/c nude mice (5–7 weeks of age) were purchased from Chongqing Byrness Weil Biotechnology Co., Ltd. (Chongqing, China). Each animal was housed under standard conditions (21 ± 1 °C, 50–10% relative Life Sciences 219 (2019) 20–30
humidity, 12 h light/dark cycle) and had free access to food and water. A549 Sorafenib at 70–80% confluence were harvested with trypsin/EDTA, washed with PBS, and resuspended in serum-free DMEM medium. Cells (1 × 107) were inoculated in the right flank of each mouse sub-cutaneously. Within days after inoculation, the tumor bearing mice were randomly assigned into 4 treatment groups (3 mice/group) of vehicle, CPT (5 mg/kg), CAA45 (5 mg/kg) and CAA45 (10 mg/kg), and treated with specific doses of compound (s) by intramuscular injection twice a week. Tumor volume and body weight of each mouse were recorded twice a week. The tumor volume (mm3) was calculated as follows: volume = (shortest diameter)2 × (longest diameter) / 2. Mice were sacrificed by intraperitoneal injection of pentobarbital sodium (1%) in 42 days after medication. Then, tumor samples were harvested, formalin-fixed, subjected to hemotoxylin and eosin (H&E) staining for evaluating histological morphology under fluorescent microscopy.
2.14. Molecular docking
Molecular docking was performed using a Sybyl-X 2.0 software. The crystal structure of Topo I protein was downloaded from the Protein Data Bank (PDB ID: 1T8I). The crystal structure of Topo I was optimized with H added and charge added by AMBER7 FF99 method. The struc-tures of small molecular database were subjected to the polar H adding and being energy optimized with a tripos force field and charged op-timized with Gasteiger-Huckel method.
2.15. Statistical analysis
Data were expressed as the mean ± SEM, and p < 0.05 was considered statistically significant. Student's t-test was used to compare means between two groups, except tumor growth. Inhibition of tumor growth was analyzed by one-way ANOVA and LSD method. All analysis was performed by using the SPSS (V19.0, SPSS Inc., Chicago, IL, USA).
Calothrixin A was reported as a weak Top I inhibitor. As a close analogue of Calothrixin A, CAA45 was expected to inhibit Top I. As shown in Fig. 2A, CAA45 caused a significant inhibition of Topo I ac-tivity in a concentration-dependent manner. Especially, the inhibitory activity of CAA45 at 10 μM outperformed CPT at the same concentra-tion, suggesting that CAA45 possessed a stronger potency in Topo I inhibition as compared to the CPT. We then docked CAA45 into the Topo I binding pocket, and found that CAA45 formed direct hydrogen bonds interaction with Asn352 and Arg364 (Fig. 2B and C), which played crucial roles for Topo I's catalytical activity. These interactions could potentially explain the inhibitory activity of CAA45 on Topo I biological function.
3.2. CAA45 reduced cancer cell growths in vitro
To test the anti-proliferative activity of CAA45 on A549, NCI-H1650, BEAS-2B and L-02 cells were treated with various concentra-tions of CAA45 for 72 h and then the cell viability was evaluated using the CCK-8 kit. As shown in Fig. 3A, CAA45 exhibited promising antic-ancer activities on the two lung cancer cell lines A549 and NCI-H1650 with IC50 values of 110 and 230 nM, respectively, where DOX and CPT displayed weaker anti-proliferative activities with IC50 values of 460 and 320 nM against A549, and 480 and 320 nM against NCI-H1650, respectively. Interestingly, CAA45 exhibited superior selectivity indexes (SI) than CPT and DOX towards the A549 and NCI-H1650 cells over the two non-cancer cells tested (Fig. 3B). Especially, CAA45 showed a SI value of 8.52, outperformed CPT and DOX with SI values of 2.67 and 2.34 towards the A549 over L02, respectively. From Fig. 3C and D,
Fig. 2. (A) Eﬀect of CAA45 on relaxation of supercoiled pBR322 DNA mediated by Topo I. Supercoiled DNA (lane DNA) incubated with Topo I in the absence (lane topo I) or in the presence of test compound at the indicated concentrations. CPT was used as a positive control. Lane 1, supercoiled plasmid DNA; lane 2, supercoiled plasmid DNA + Topo I; lane 3, supercoiled plasmid DNA + Topo I + CPT (10 μM); lanes 4–6, supercoiled plasmid DNA + Topo I + CAA45 (1, 5, and 10 μM). (B and C) Molecular docking of CAA45 with the Topo I (PDB code: 1T8I). Docking model of CAA45 in the binding pocket (B); stereo view of CAA45 in the binding pocket
(C). Amino acid residues and compound CAA45 are shown as stick models, H-bonds are shown as yellow dashed lines. The 3D graphical presentations were drawn by PyMol. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)