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  • br Fig E ect of CAA on

    2020-08-12


    Fig. 9. Effect of CAA45 on autophagy in A549 cell. (A) Cells were plated on coverslips in 24-well plates, treated with CAA45 (0, 0.06, 0.12, 0.25 μM) or Rapa (0.30 μM) for 12 h, then stained with 0.05 mM MDC for 30 min at 37 °C. After washing with PBS, the stained A549 774594-96-6 were immediately examined by Leica TCS Sp8 confocal microscope. Organelles stained with bright blue fluorescence indicate the autophagic vacuoles. Scale bar: 25 μm. (B) After treatment with CAA45 (0, 0.06, 0.12, 0.25 μM) for 12 h, cell lysates were prepared for western blot. The autophagy marker protein LC3-B were detected as described in the Materials and methods section. (C) A549 cells were transfected for 6 h with Ad-mCherry-GFP-LC3B adenovirus at an MOI of 20 at 37 °C, then treated with 1% DMSO, CAA45 or Rapa for 12 h. The treated cells were fixed to be available for microscopy. The GFP/mCherry images were acquired using Leica TCS Sp8 confocal microscope. Scale bar: 5 μm. Rapa: rapamycin. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    increased in A549 cells, suggesting that CAA45 induced autophagy in A549 cells. As expected, rapamycin also increased the number of au-tophagic vacuoles under the same conditions. To further evaluate the influence of CAA45 on autophagy, we transfected A549 cells with Ad-mCherry-GFP-LC3B adenovirus. As shown in Fig. 9C, only signals of GFP and mCherry protein which re-present diffuse LC3 protein were found in the cytoplasm before treat-ment of CAA45. After treatment with CAA45 at 0.06, 0.12, and 0.25 μM for 12 h, the mCherry and GFP dot signals significantly increased in A549 cells, suggesting that CAA45 induced autophagic vacuole for-mation.
    Finally, we determined the effect of CAA45 on the expression of LC3B, an autophagy marker protein. As shown in Fig. 9B, after treat-ment with CAA45 for 12 h, the conversion of LC3B I to II increased in a 
    concentration-dependent manner, further demonstrating the autop-hagy-inducing activity of CAA45.
    3.6. CAA45 induced Akt inactivation, JNK activation and up-regulation of
    It has been reported that the PI3K/Akt pathway, MAPK (JNK) sig-naling and p53 play important roles in cell proliferation, survival, apoptosis and autophagy. However, the relationship between these signaling pathways and the effect of CAA45 on proliferation, migration and autophagy of lung cancer cells has not yet been reported. Our study aimed to explore the potential effect mechanisms. As shown in Fig. 10A, the expressions of p-AKT in A549 cells after treating with CAA45 were significantly reduced (p < 0.01). In addition, the phosphorylation of
    Fig. 10. CAA45 induced Akt inactivation, JNK activation and up-regulation of p53 expression in A549 cells. Cells were treated with DMSO or CAA45 for 24 h. Phosphorylated Akt (p-Akt), Akt, phosphorylated JNK (p-JNK), and JNK, p53 expressions were detected by western blot. The values obtained represent the mean ± SEM for three separate experiments. * = p < 0.05 vs control, ** = p < 0.01 vs control.
    3.7. CAA45 inhibited tumor growth in vivo
    The in vivo antitumor activities of CAA45 were evaluated in a nude mouse xenograft model. As shown in Fig. 11A, CAA45 led to a sig-nificant reduction in tumor growth as compared to the untreated con-trols for 42 days following drug exposure (p < 0.01). To evaluate whether CAA45 treatment might result in morphological changes in A549 cells in vivo, excised tumor samples were sectioned and stained with H&E. H&E staining of sections of the tumors from mice treated with CAA45 exhibited significant changes in morphology, with signs of apoptotic cells (Fig. 11C). However, compared to the vehicle group, slight body weight gain (Fig. 11B) was observed among treat groups. The slow body weight gain suggested that CAA45 might have some toxicity, similar to the control drug CPT.
    4. Discussion
    We synthesized a potent CAA analogue CAA45, fully evaluated its anti-lung cancer activity, and explored its mechanism of action. This study is the first report that CAA45 inhibits human non-small cancer cell growth in vitro and in vivo. CAA45, a Topo I inhibitor, is found to cause cell cycle arrest in S phase, induce cell apoptosis and autophagy, and inhibit cell migration in A549 cells. Furthermore, we found that the 
    pro-apoptotic effect of CAA45 was regulated by mitochondria mediated apoptosis pathway. Both CAA45 induced apoptosis and autophagy were regulated by Akt/JNK/p53 signaling pathway.
    Topo I is a promising target for developing anti-cancer drugs. Our data demonstrated that CAA45 strongly inhibited Topo I activity at 10 μM, outperforming CPT under the same experimental conditions. Furthermore, CAA45 induced cell cycle arrest in the S phase in A549 cells, indicating intracellular DNA damage. Based on the above ob-servations, we could conclude that inhibition of Topo I enzyme by CAA45 contributed to the DNA damage, resulting in cell cycle arrest at S phase and inhibition of cell proliferation.