br CAA inhibited A and NCI H cell proliferation in
CAA45 inhibited A549 and NCI-H1650 cell proliferation in concentra-tion-dependent and time-dependent manners. In addition, CAA45 re-pressed the colony formation of A549 and NCI-H1650 cells. As shown in Fig. 3E and F, CAA45 clearly inhibited most of A549 and NCI-H1650 cell colony formation at 0.06 μM, and even at 0.01 μM, CAA45 was able to partially reduce its colony formation in both cancer cell lines. Moreover, A549 and NCI-H1650 cells formed fewer colonies with in-crement concentration of CAA45 in a dose response manner. All these results indicated that CAA45 exhibited a nanomolar anti-lung cancer activity, yet low toxicity against the normal human cell line.
Since cell migration was observed in cell growth, we therefore in-vestigated the eﬀect of CAA45 on cell migration in A549 cells. The results of wound healing and transwell assays showed that A549 cell migration was significantly reduced (p < 0.05) after treating with CAA45 at 0.05, 0.10, and 0.15 μM as compared with the control group (Fig. 4A), suggesting that CAA45 strongly inhibits cancer cell migra-tion. To understand if the reduced cancer cell migration is related to the protein matrix metalloproteinase-2 (MMP-2) and matrix
metalloproteinase-9 (MMP-9), which degrade components of the ex-tracellular matrix and thus play a pivotal role in cell migration during physiological and pathological processes, we next explored the eﬀect of CAA45 on MMP-2 and MMP-9 expressions in A549 cells. As shown in Fig. 4B, the protein expression level of MMP-2 was significantly de-creased after treating with CAA45 at 0.06, 0.10 and 0.15 μM. Similarly, MMP-9 protein expression was also significantly reduced with the treatment of CAA45 (0.15 μM). These results demonstrated that CAA45 inhibited A549 cells migration, partially ascribe to the reduction of the MMP-2 and MMP-9 expressions.
3.4. CAA45 induced A549 Phosal50PG arrest in S phase and apoptosis
To explore the mechanism of cancer cells death, CAA45 was used to induce A549 cells apoptosis, which was then examined with Annexin V-
Fig. 3. Response of lung cancer and non-cancer cell lines to CAA45. (A) Cytotoxicity of CAA45 against lung cancer and non-cancer cell lines. Data are the mean ± SEM for 3 independent experiments. (B) The selectivity index was calculated from the IC50 values of non-cancer cell lines L02 or BEAS-2B dividing by the IC50 values of either A549 or NCI-H1650 cells. Selectivity index > 1 denotes preferential activity against cancer as compared to non-cancer cells, whereas the value < 1 denotes equivalent or reduced activity against cancer cells as compared to non-cancer cells. (C) Full dose responses of CAA45 inhibiting A549 and NCI-H1650 cell proliferation. (D) CAA45 time dependently inhibited A549 and NCI-H1650 cell growth. (E, F) CAA45 inhibited A549 and NCI-H1650 cell colony formations.
Fig. 4. CAA45 inhibited A549 cells migration. A549 cells were treated with DMSO or CAA45 (0.05, 0.10, and 0.15 μM) for 24 h. (A) The upper photo showed the closure of A549 cells after being scratched (×100) for 24 h, while the below showed the crystal violet stained A549 cells, which migrated to lower chamber after transwell (×100) for 24 h; (B) Western blot analysis of MMP-2 and MMP-9 expression in A549 cells. Bar graph represents mean ± SEM of the MMP-2 and MMP-9 expression level, which were derived from the optical density (OD) of immune reactive band analyzing by image J software. Each determination was done at least in triplicate. * = p < 0.05 vs control, ** = p < 0.01 vs control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Induction of cell cycle arrest and apoptosis by CAA45 treatment. A549 cells were treated with DMSO or CAA45 at diﬀerent concentrations for 24 h. (A) Cells were stained with PI and cell cycle distribution was analyzed by using a flow cytometer. (B) For apoptosis, cells were stained with Annexin V-FITC/DAPI and analyzed using a flow cytometer. The values represent the mean ± SEM for three separate experiments. * = p < 0.05 vs control, ** = p < 0.01 vs control.
Furthermore, protein immunoblotting data showed that the ex-pression level of cytochrome c in cell cytoplasm was increased gradu-ally by treating with increased concentrations of CAA45 (0.06, 0.12, 0.25 μM) (p < 0.05 or p < 0.01), while significantly decreased (p < 0.01) in mitochondria (Fig. 6). These results indicated that CAA45 promoted the release of cytochrome c from mitochondria into cell cytoplasm.
To further explore the mechanism by which CAA45 induced apop-tosis, we determined the protein expressions of C-caspase-3, C-caspase-8, C-caspase-9, Bad, Bax, Bcl-2 and Bcl-xl in A549 cells. As shown in
MDC incorporation assay is an autofluorescent substance which selectively accumulates in acidic vesicular organelles (AVOs), and therefore is used as a marker for autophagy. As shown in Fig. 9A, after treatment with CAA45, the number of autophagic vacuoles was