一、 NSC746364 suppresses cell proliferation of A549 human lung cancer cell line
The effect of NSC746364 on cell proliferation was assessed using the MTT proliferation assay. To test the effect of NSC746364 on the proliferation of A549 cells, the cells were treated with different concentrations of NSC746364 (0 μM, 5 μM, 10 μM, 20 μM). After 24 h (A) and 48 h (B) incubation, the cell viability was determined by using the MTT assay. As shown in Figure 11 A and B, treatment with NSC746364 significantly inhibited the viability and proliferation of cells, and these effects occurred in a dose-dependent manner.
Figure 11 Cell survival inhibition of A549 human lung cancer cell lines by NSC746364 assessed using the MTT proliferation assay. NSC746364 significantly suppressed proliferation of A549 cells in concentration dependent manner. Cells were treated with NSC746364 at concentrations 5, 10, and 20 μM for 24 h (A) and 48 h (B). Untreated groups (0 μM) contained DMSO less than 0.01％. All results were obtained from at least 3 times independent experiments. Statistical significance was determined using
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ANOVA test, (* p<0.05).
二、 NSC746364 modulates cell cycle progression
The consequences of telomere dysfunction caused by telomere-disrupting agents, such as G-quadruplex inhibitors, resulting in activation of DNA damage response signaling and leading to activation of cell cycle checkpoints [161, 205-208]. The cell cycle distribution of A549 cells was examined by flow Cytometry on cells treated with various concentrations (0 μM, 5 μM, 10 μM, 20 μM) of NSC746364 for 24 h Figure 12 (A to E) and 48 h (F to J). In the present study, flow cytometric analysis showed that NSC746364 modulated cell cycle progression through inducing cells to accumulate at G2/M-phase with concurrent decrease of cells at G0/G1 and S phase.
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Figure 12 Flow cytometric analyses elucidated the effect of NSC746364 on the cell cycle phase distribution in A549 cells. Averages from three independent experiments of 0.01% DMSO-treated cells (0 μM, as a control) and NSC736364-treated cells at doses 5, 10 and 20 μM for 24 h (A to E) and 48 h (F to J) were calculated. Statistical significance was analyzed using ANOVA test, (* p<0.05). NSC746364 induced G2/M cell cycle arrest in a dose-dependent manner.
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三、 NSC746364 modulates cell cycle progression through activation of DNA damage sensing pathways
To further elucidate the mechanism by which NSC746364 induced G2/M arrest through investigating G2/M cell cycle regulatory proteins. Two dominant regulatory proteins, Cyclin B1 and the mitotic marker protein kinase (Cdk1/Cdc2) were determined by western blot analysis. We found that NSC746364 down-regulated Cyclin B1 levels in a dose-dependent manner. However, the protein levels of Cdc2 were not affected by NSC746364. We also found that NSC746364 up-regulate both Chk1 and Chk2 phosphorylation at Ser-345 and Thr-68, respectively. However, NSC746364 markedly increases Chk1 phosphorylation at Ser-345 but not Chk2 phosphorylation at Thr-68. Furthermore, the significantly increased Chk1 phosphorylation at Ser-345 could be detected very early after 15 min of NSC746364 treatment.
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Figure 13 Effects of NSC746364 on protein expression level of Cyclin B1, p-Chk1, and p-Chk2 in A549 cells. (A) Cells were treated with 5, 10 and 20 μM for 24 h. Cell lysates were analyzed by Western blotting with anti-Cyclin B1, anti-Cdc2, and anti-β-actin antibodies. The bar graphs are results of densitometry analyses of the ratio of Cyclin B1 to β-actin. (B) A549 cells were incubated with NSC746364 for 0, 15, 30, and 60 minutes. The protein levels of p-Chk1, p-Chk2, Chk1, Chk2, and β-actin were determined by Western blot. The bar graphs are results of densitometry analyses of the ratio of p-Chk1 to β-actin and p-Chk2 to β-actin. Each value represents the mean±
S.D. *P<0.05, as compared to the control (0 μM and 0 min), n=3.
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四、 NSC746364 induces A549 cells apoptosis
As shown in our MTT results, tumor growth was strongly suppressed by NSC746364 treatment, suggesting the cytotoxic effects of NSC746364 on lung cancer cells. To test this hypothesis, we examined the potential apoptotic signaling pathways in A549 cancer cell lines. Our results show that NSC746364 induced tumor cell apoptosis by activating Caspase-3 (Figure 14A). The increasing intensity of DAPI staining demonstrates the dose dependent apoptotic effects of NSC746364 on A549 cells (Figure 14B).
Furthermore, Annexin V-FITC/propidium iodide (PI) staining was another alternative way to evaulate apoptosis. As shown in Figure 14C, NSC746364 significantly increased PI-positive and Annexin-V-positive cells in a dosel–
dependent-manner.
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Figure 14 NSC746364 induces cell apoptosis. (A) A549 cells were incubated with NSC746364 at concentration 5, 10 and 20 μM for 24 h. Cell lysates were analyzed by Western blotting with anti-Caspase-3 and anti-β-actin antibodies.
The bar graphs are results of densitometry analyses of the ratio of either pro-Caspase-3 to β-actin or active Caspase-3 to β-actin. (B) The cells were cultured in 10% FBS with or without NSC746364 for 24 h. After 24 h, the cells were fixed and incubated with DAPI for 1 min, and observed using a fluorescent microscope. White arrows indicate the apoptotic nucleus. All graphs were taken at 40X. (C) Apoptosis of A549 cells treated with different concentrations of NSC746364 (0, 5, 10 and 20 μM) for 24h was assessed using Annexxin V/PI staining and flow cytometry. Cells in the lower right quadrant (Q4) indicate early apoptotic cells. Cells in the upper right quadrant
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(Q2) indicate late apoptotic cells.
五、 ATM/ATR-Chk1/Chk2 DNA damage sensing signaling pathways are responsible for NSC746364’s action in A549 human lung cancer cell lines We have previously demonstrated that cells treated with NSC746364 showed activated Chk1 and Chk2 protein phosphorylation levels. To further confirm the mechanism that underlies the antitumor effects of NSC746364 via ATM/ATR DNA damage sensing pathways, we examined the effect of caffeine, an inhibitor of ATM and ATR kinases [209], on tumor cells. Cells were either incubated with caffeine (10 mM) for 15 min or treated for 15 min before NSC746364 (20μM) treatment. As the data indicated (Figure 15), cells treated with NSC746364 significantly accumulated in G2/M phase. In contrast, pre-treated cells with caffeine markedly attenuated the effect of NSC746364 on cell cycle regulation. Furthermore, caffeine also significantly increased the cell viability as compared to NSC746364 alone group.
Figure 15 ATM and ATR blockage attenuated the effect of NSC746364 on cell cycle regulation and cell viability. Cells were pre-treated with caffeine (10 mM) for 15 min, following incubated with NSC746364 (20μM) for 24 h. Cell cycle phase distribution (A) and cell viability (B) were determined by flow
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cytometry and MTT assay, respectively. Each value represents the mean±S.D.
*P<0.05, as compared to the control (0 μM). #P<0.05, as compared to the NSC746364 treated group, n=3.
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