Arsenic trioxide (ATO; As2O3) has anti-cancer effects in various solid tumors as well as hematological malignancy

Arsenic trioxide (ATO; As2O3) has anti-cancer effects in various solid tumors as well as hematological malignancy. death induced by ATO/VPA. In the xenograft model, ATO/VPA synergistically inhibited growth of NCI-H460-derived xenograft tumors. In conclusion, the combination of ATO/VPA effectively inhibited the growth of lung cancer cells through G2/M-phase arrest and apoptotic cell death, and had a synergistic antitumor effect in vivo. 0.05 as compared with the control group. (= 3). 2.2. Effects of ATO and VPA Alone and K02288 small molecule kinase inhibitor in Combination on Cell Morphology and Cell Cycle Distributions Morphologic changes were visualized by inverted microscopy for NCI-H460 and NCI-H1299 cells that were treated with 3 M ATO and 3 mM VPA alone and in combination after 72 h incubation. The concentrations of 3 M ATO and 3 mM VPA and the incubation time of 72 h were considered as suitable doses and time to distinguish the differences of dead and live cells. Compared with groups treated with single drugs only, a decrease in cell number was observed after treatment with ATO/VPA (Figure 2A). Growth inhibition can be explained by an arrest during cell cycle progression, therefore cell cycle distributions were also analyzed at 72 h (Figure 2B). While 3 M ATO induced G2/M-phase arrest of the K02288 small molecule kinase inhibitor cell cycle in both NCI-H460 and NCI-H1299 cells, 3 mM VPA induced G1-phase arrest in NCI-H1299 cells (Figure 2B,C). In addition, ATO/VPA significantly increased the proportions of G2/M-phase cells in both NCI-H460 and NCI-H1299 cells (Figure 2B,C). Open in a separate window Figure 2 Effects of ATO and VPA alone and in combination on cell morphology and cell cycle distributions. Exponentially growing NCI-H460 and NCI-H1299 cells were treated with 3 M ATO and/or 3 mM VPA for 72 h. (A): Rabbit Polyclonal to MMP27 (Cleaved-Tyr99) Cell morphology changes were captured by an inverted microscope (40). (B): Cell cycle distributions were measured by BD Accuri C6 flow cytometry (M1 regions show sub-G1 cells, M2: G1 phase, K02288 small molecule kinase inhibitor M3: S phase, M4: G2/M phase). (C): Percentages of G1, S, and G2/M phases in M2, M3, and M4 regions of Figure 2B. (D): Percentages of sub-G1 cells in M1 regions of Figure 2B. (E): LDH release in NCI-H460 and NCI-H1299 cells co-treated with ATO/VPA. * 0.05 as compared with the control group. # 0.05 as compared with cells treated with ATO or VPA. (= 3). 2.3. Effects of ATO and VPA Alone and in Combination on Cell Death, LDH Release, and Apoptosis ATO/VPA (3 M ATO and 3 mM VPA) significantly increased the percentages of sub-G1 cells in both NCI-H460 and NCI-H1299 cells (Figure 2D). LDH release was not increased in NCI-H460 and NCI-H1299 cells after treatment with ATO/VPA (Figure 2E). Whether ATO and VPA induces apoptotic cell death in cells was evaluated using annexin V-FITC/PI staining cells. Treatment with 3 mM VPA significantly increased the number of annexin V-staining cells in NCI-H460 cells whereas 3 M ATO augmented the number in NCI-H1299 cells (Figure 3A,B). The percentages of annexin V-staining cells in NCI-H460 and NCI-H1299 cells treated with ATO/VPA were synergistically increased, compared with cells treated with single drugs alone (Figure 3A,B). K02288 small molecule kinase inhibitor In addition, changes in apoptosis-related proteins were detected with Western blotting. The intact form of PARP was clearly reduced in both NCI-H460 and NCI-H1299 cells after treatment with ATO/VPA, and the cleavage form of PARP was strongly induced in these cells. The cleavage forms of caspase-3, caspase-8, and caspase-9 also clearly increased in ATO/VPA-treated cells, compared with cells treated with single drugs alone (Figure 3C). Open in a separate window.