Sodium Butyrate Overrides The Resistance Of NB4 Cells To Arsenic Trioxide

Objective:Arsenic trioxide (As2O3) is an arsenic derivative, which exhibits potent growth inhibitory effects against malignant cells. As2O3 is highly effective in treatment of patients with refractory acute promyelocytic leukemia (APL). With the investigation have been further progressed, the resistance to As2O3 was observed in many tumor cells. Chemotherapeutic treatment leads to the activation of conflicting signaling pathways that both promote and inhibit cell death. The resistance to chemotherapeutic drugs might be related with activation of signaling pathways about inhabitation of cell growth and apoptosis. However, whether other mechanism of resistance exists is totally unknown.The mechanism by which arsenic triggers apoptosis remains controversial. There is evidence that it down-regulates Bcl-2 protein and activates caspase-3-like caspase activity. Some reports have shown that arsenic-induced apoptosis is caused by a direct effect on the mitochondrial permeability transition pore, resulting in loss of the mitochondrial transmembrane potential. Other studies have demonstrated that arsenic compounds can disrupt mitosis and therefore induce apoptosis in a variety of cell systems. The disruption of mitosis was shown to be due to interference with tubulin polymerization and disruption of mitotic spindles. However, arsenic not only induces apoptosis in tumor cells, but also arrests tumor cells in G2/M phase of cell cycle. Until now,the relationship between disruption of cell cycle and apoptosis induced by chemothrapuetic drugs is not completely understanded.In this study, we have analyzed the srelation among the G2/M arrest, apoptosis, and resistance to As2O3 in NB4 cells treated with As2O3 and gain primary insight into the possible mechanism. Materials:1. Cell lineAcute promyelocytic leukemia cell line NB4 was kindly provided by the department of experimental medicine, General Hospital of Shenyang military command.2. Main reagents: As2O3, RPMI 1640 media, sodium butyrate(NaB), fetal calf serum, agarose, prolease K, RNase,Propidium Iodide(PI).3. Major instruments: Labofuge 400R centrifuge, FACSort flow cytometer, BB16/BB5060 CO2 incubator, electrophoretic apparatus, and Lamda 2S ultraviolet spectrophotometer.Method:NB4 cells treated with different concentration of As2O3 from 0.5 to 4.0μM were harvested at hour 16 or NB4 cells treated with 2μM were harvested at different points of time from hour 4 to hour 36. In some experiments, NB4 cells were treated with As2O3 (2μM), NaB (5mM), As2O3 and NaB respectively and harvested at hour 16. The distribution of cell cycle was analyzed with FACSort flow cytometer. Apoptosis were accessed with flow cytometry or based on 3'-OH end labeling of DNA strand breaks with dUTP-FITC.DNA fragmentation were measured by agarose gel electrophoresis of genomioc DNA form the treated cells.Result:Experimental Results1. After being treated with As203 at the concentration of 0, 0.4, 1, 2, 4,and 8μM for 16 hours, the proportion of NB4 cells in G2/M phase of cell cycle increased form 15.10±4.33% at 0μM to 42.78±4.89% at 4μM. The apoptosis of the treated-NB4 cells increased with increment of the concentration of As2O3 as well. These data suggested that As2O3 induced G2/M phase arrest and apoptosis of NB4 cells in dose response manner. However, the concentration at which apoptosis was induced is higher than that at which the cells were blocked in G2/M phase.2. After being treated with As As2O3 at 2μM for different periods of time, the proportion of NB4 cells in G2/M phase of cell cycle increased form 15.10±4.33% at hour 0 to 55.34±4.95% at hour 28. The apoptosis of the treated-NB4 cells increased with prolonging of the time of treatment with As2O3 as well. These data suggested that As2O3 induced G2/M phase arrest and apoptosis of NB4 cells in time response manner. However, apoptosis was induced later than G2/M arrest was induced.3. The histone deacetylase (HDAC) inhibitor, sodium butyrate (NaB), decreased the proportion of As2O3-induced NB4 cells in G2/M phase and increased the proportion of apoptotic cells as well.Discussion:Arsenic trioxide (As2O3) is an arsenic derivative, which exhibits potent growth inhibitory effects against malignant cells. However, it had been reported that many tumor cells developed resistance to the treatment of As2O3. Microtubule-targeted agents, such as paclitaxel, through binding to b-tubulin, decrease microtubule dynamic instability, interfere with G2/M transition, induce mitotic arrest, and subsequently trigger the molecular signaling for the mitochondrial pathway of apoptosis. Arsenite has also been demonstrated to attenuate spindle dynamics and thereby induce mitotic arrest and mitosis-mediated apoptosis. However, the relationship between the cell-cycle perturbation and apoptosis induced by these anti-microtubule agents remains to be elucidated.In this study, we found that that As2O3 induced G2/M phase arrest and apoptosis of NB4 cells in dose and time response manner. The G2/M phase arrest appeared earlier in time and lower in dosage than apoptosis did. These results demonstrated that the G2/M phase arrest in NB4 cells treated with As2O3 was due to direct actiation of signaling pathways to G2/M arrest by As2O3,but not due to the activation of apoptosis signaling pathways and subsequent damage of DNA. It is well known that chemotherapeutic treatment leads to the activation of conflicting signaling pathways that both promote and inhibit cell death. If this point of view is also applied to As2O3, therefore, activation of signaling pathway of G2/M phase arrest might play an important role in the resistace of tumor cells to As2O3Due to the facts that PTX treatment resulted in an increase in the expression of cyclin B and G2/M phase arrest and the histone deacetylase (HDAC) inhibitor, sodium butyrate (NaB), overrided PTX induced-G2/M phase arrest and increased apoptosis, NaB was employed to cotreat NB4 cells with As2O3. At the concentration we used, treatment of NaB decreased the proportion of As2O3-induced NB4 cells in G2/M phase and increased apoptosis of the cells as well. This result at least further demonstrated that G2/M phase arrest in NB4 cells treated with As2O3 was due to direct actiation of signaling pathways to G2/M arrest by As2O3.Therefore, based on our results, treatment with drugs, such as arsenic, that arrest cells in G2/M phase and cause a persistent activation of M-phase kinases, should be combined with drugs that reduce cdk1 activity such as flavopiridol and the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA) to avoid the acquisition of chemoresistence. Conclusion:1. As2O3 arrested NB4 cells in G2/M phase in dose-response manner and time-response manner;2. G2/M phase arrest induced by As2O3 appeared earlier in time and lower in dosage than apoptosis;3.NaB overrided As2O3 induced-G2/M phase arrest and increased apoptosis.