The Study Of Apoptosis And ROS Production Induced By DADS In Combination With Bortezomib In Neuroblastoma Cells

Background Neuroblastoma is one of the most common pediatric solid tumor in children. At present, the overall outcome remains poor. As the emerging of drug resistance, the recurrent rate is significantly increased. Therefore the attention of researchers had been drawn to explore new therapy methods to treat the malignance and overcome drug resistance. Recently, more strategies to manipulate the apoptotic process in cancer cells are emerging as potential therapeutic tools. Meanwhile, it is found that the efficacy of sole drug is always limited and drug resistance is developed easily. Drugs with different mechanisms combinations have been used for treating diseases and reducing suffering, which has drawn much attention. Diallyl disulfide (DADS), an oil-soluble allyl sulfur compound found in processed garlic, was found to inhibit the growth of various tumors by decreasing the cell growth rate, inducing apoptosis and cell cycle arrest and et al. One of the important mechanisms is to induce ROS (reactive oxygen species) production, which resultes in the activation of the JNK/c-Jun transduction pathway, then DADS induces apoptosis through the activation of the mitochondrial pathway. Bortezomib, a boronic dipeptide, is a potent proteasome inhibitor that selectively and reversibly inhibits proteasome, inducing the degradation of ubiquitynated proteins critically involved in the regulation of apoptosis, growth arrest and signaling pathways. It has been proposed Bortezomib increases ROS producton by inhibiting NF-κB activity, which is a therapeutic target for exerting antiapoptotic and antiproliferative functions. Inducing stimuli trigger transcription factor NF-κB translocating to the nucleus, where it activates gene expression, which protects tumor cells from apoptosis and oxidative stress. In short, DADS induces ROS production, which results in the activation of the JNK/c-Jun transduction pathway, on the other hand, bortezomib inhibits NF-κB activity, indirectly increases ROS production. We, therefore, combined DADS and bortezomib to strengthen the therapeutic effect.Methods Following DADS and bortezomib alone and in combination treatment for 24 h, cell proliferation was determined by MTT assay, cell apoptosis was assayed by flow cytometry and stained with Hoechst-3352, the effect on ROS was analysed by flow cytometry.Results To explore the effect of DADS and bortezomib alone and in com-bination on cellular proliferation in SH-SY5Y cells, the cells were treated with 100μmol/L DADS,10 nmol/L Bortezomib alone and in combination for 24 h. The results showed that DADS and bortezomib can inhibit SH-SY5Y cell proliferation. Each drug alone was able to induce a dose-dependent inhibition of cell proliferation, with a significant enhanced antiproliferative effect for the drugs used in combination.To confirm the effects of DADS and bortezomib alone and in combination on cellular apoptosis in SH-SY5Y cells, the cells were treated with 100μmol/L DADS and 10 nmol/L bortezomib alone and in combination for 24 h. Phenotype of SH-SY5Y cells after stained with Hoechst33528 was observed. We found the apoptotic chara-cter of the SH-SY5Y cells:cyctoplasmic and nuclear membrance held integrity and chromosomes condensed which represented the final steps of apoptosis, which showed DADS and bortezomib alone was able to induce apoptosis; Flowcytometry also demonstrated that DADS and bortezomib alone was able to induce apoptosis, with an increased apoptosis for DADS and bortezomib used in combination (P<0.01).To investigate the role of ROS of DADS and bortezomib alone and in combina-tion during cellular apoptosis in SH-SY5Y cells, the cells were treated with 100μmol /L DADS and 10nmol/L bortezomib alone and in combination for 12 h. Flowcyto-metry demonstrated that an increase in ROS generation was observed when cells were exposed to DADS and bortezomib alone and a significant increase in ROS generation was detected when DADS was combined with bortezomib (P<0.01).Conclusion Our researchers suggested that bortezomib can facilitate the apoptosis of neuroblastoma cells induced by DADS. The enhanced production of ROS maybe play an important role during this process.