Mechanism Of Cell Cycle Arrest And Apoptosis Of NB4 Sodium Selenite By ROS / JNK / ATF2 Pathway Induced Leukemia

The essential trace element, selenium, is of fundamental importance to human health and the prevention of tumors. A lot of experimental evidences showed that ultra doses of selenium could effectively induce tumor cells apoptosis, including prostate cancer, bladder cancer, thyroid cancer, esophageal cancer, liver cancer, colon cancer, lung cancer and leukemia. Leukemia, also known as blood cancer, is a kind of hematopoietic cells malignant hyperplastic diseases, the mortality is high and the current treatment is not satisfactory. Therefore, making a thorough inquiry into the molecular mechanisms of selenium induced leukemia cell apoptosis would be of great significance for the development of drugs containing selenium and the treatment of leukemia with new ideas and theoretical basis.In this study, selenite acted as an antitumor drug to induce apoptosis of leukemia cells and studying the unknown mechanisms responsible. First of all, we clarified that selenite caused NB4cell apoptosis in a time-dependent manner by flow cytometry and TUNEL assay. At the same time, we found that the DNA synthesis was also significantly reduced in a time-dependent manner. Therefore, after treatment as indicated time with selenite, cells were evaluated by PI staining and further analyzed with flow cytometry. We noted that selenite arrested NB4cells at the G0/G1phase. Additional experiments had confirmed some cell cycle related protein altered in line with this arrest. Moreover, inhibition of JNK/ATF2pathway was also observed. These results suggested that selenite-induced apoptosis might be related to the alteration of cell cycle progression and the inhibition of the JNK/ATF2axis.In the next step of investigation, we discovered that the reactive oxygen species raised rapidly after selenite exposure. Both of the apoptosis and cell cycle arrest caused by selenite were reversed while combinational uses of an active oxygen scavenger MnTMPyP with selenite. Western blotting indicated that selenite-induced decreases of cyclin D1, cyclin D3, CDK4, cyclin A and phospho-Rb were canceled because of MnTMPyP pre-treatment, while the selenite-induced activation of caspase-3and PARP were also abolished in this condition. Meanwhile, scavenge of ROS also rescued the inhibition of the JNK/ATF2axis. Notably, cells that were treated with H2O2that were used as a positive control could also result in the cell cycle arrest, apoptosis induction and JNK/ATF2axis inhibition.The subsequent experiments further proved that the JNK interacted with ATF2 through IP experiment and indirectly immunofluorescence assay. Through siRNA interference, the uses of JNK specified inhibitor and the JNK over-expression plasmids transfection experiments, we concluded that the ROS-induced JNK-related pathway might be responsible for the selenite-induced apoptosis and cell cycle arrest. Moreover, ChIP assay further indicated that ATF2, the downstream of JNK, bound to the promoters of cyclin D3, cyclin A and CDK4which was attenuated by selenite. Additional transfection experiments of siRNA targeting ATF2and ATF2over-expression plasmids had confirmed the inactivation of ATF2might be the reason by which selenite induced apoptosis and inhibited the expression of cyclin D3, cyclin A and CDK4. Therefore our experimental results confirmed that the inhibition of JNK/ATF2pathway under the action of active oxygen increases was responsible for the decreases of eel cycle related proteins and the increases of apoptotic markers.Finally, we established a leukemia tumor cells bearing nude mice model. A TUNEL assay and H&E staining indicated that selenite-treated tumor tissues exhibited more dead cells than tissues of untreated mice. As CD33was a leukemic disease marker, we indirectly labeled CD33-positive cells in spleen and liver tissue sections. Immunohistochemical staining indicated that there were many CD33-positive cells in tissues of control group and that these cells were decreased after selenite treatment. These results suggested that selenite had a therapeutic effect in vivo. We also investigated whether mechanisms of selenite activity in vivo are consistent with those that we described in vitro. Whole tissue cell lysates are extracted, and Western blot analysis also confirmed that the JNK/ATF2axis, cyclin D3, CDK4, cyclin A, phospho-Rb, PARP and caspase-3were altered in a similar manner as previously described in vitro. Finally, these proteins were indirectly labeled with their primary antibodies, and immunohistochemical staining results also demonstrated that these proteins were altered in the same manner as shown in vitroThrough this study, we clarified the mechanisms of selenite-induced tumor cells apoptosis after blocking cell cycle process. Reactive oxygen species was the upstream of all of these events. It provided a good theoretical basis for selenite clinical applications. In addition, we confirmed the sodium selenite could not only induce tumor cell apoptosis, also inhibit tumor cell invasion and metastasis at the animal level. It supported that selenite had potential values in the treatment of leukemia.