The Research Into BSO-mediated Oxidative Stress Combined With Cisplatin Promoting The Apoptosis Of Human Lung Adenocarcinoma A549Cell Spheres

Background and purpose:Currently, non-small cell lung cancer (NSCLC) mortality has shown anincreasing trend year by year, ranking first in the death of cancer. And tumor tissue oflung cancer is highly resistant to diamminedichloroplatinum (DDP), resulting in thefailure of chemotherapy, which has become a clinical subject to be addressedurgently.The “cancer stem cell hypothesis” theory is that cancer stem cells（CSCs) arefew of tumor cells with stem cell properties group and that the primitive cells notfully differentiated yet possess the potential of self-renewal and multi-directionaldifferentiation. A single CSCs with tumorigenicity may be initiating cells of tumorrecurrence. Based on our studies have successfully isolated lung adenocarcinomaCSCs. Compared with non-CSCs, lung adenocarcinoma CSCs have their uniquefeatures of energy metabolism such as low mitochondrial DNA copy number, lowconcentration of intracellular reactive oxygen species (ROS), low glucose and lowoxygen consumption. Besides, it is considered that maintaining a low intracellularROS concentration within their CSCs is closely related to self-renewal, differentiationand high tumorigenicity. In view of the important role of ROS by radiotherapy inpromoting tumor apoptosis, we believe that the intracellular ROS concentration ofCSCs may also affect the efficacy of tumor chemotherapy drugs, and that promotingROS accumulation by combination chemotherapy in the CSCs may become a newstrategy for comprehensive lung cancer therapy.Therefore, human lung adenocarcinoma A549cells were cultured withserum-free medium (SFM) to A549cell spheres in this study to investigate thedifference of glutathione-S-transferase-π (GST-π) expression and intracellular ROSconcentration between A549cells and A549cell spheres. Meanwhile, we respectivelygave control group、 DDP group、 combined GST-π inhibitorDL-buthionine-S,R-sulfoximine (BSO) and N-acetylcysteine (NAC) with DDPintervention to contrapose the two kinds of cells at the same time and simultaneously detect the concentration of intracellular ROS in each group and their apoptosis.Objectives:1、Compare of ROS concentration within different forms of a single A549cellcloning.2、Compare the difference between A549cells and A549cell spheres in theexpression of GST-π, concentration of intracellular ROS and apoptotic rate after theintervention of combinations of different drugs and simultaneously discuss theircorrelation.Methods:1、Fluorescence microscope was used to detect ROS fluorescence in differentforms of a single A549cell cloning.2、Western Blot was used to detect the expression of GST-π in A549cells andA549cell spheres,24hours after the intervention of combinations of different drugs.3、Flow cytometry was used to probe the concentration of intracellular ROS andapoptosis of A549cells and A549cell spheres,24hours after the intervention ofcombinations of different drugs.Results:1、The intracellular fluorescent ROS in A549cells holoclone was significantlylower than the paraclone（24.13±1.79vs77.89±6.71P<0.01）.2、The expression of GST-π protein in A549cell spheres was higher than A549cells （1.45±0.07vs1.25±0.06P<0.05）, while maintaining low intracellularconcentrations of ROS were maintained (272.67±16.04vs326.33±12.34P<0.01).After the intervention of DDP, the concentration of intracellular ROS in A549cellspheres rose smaller（585±12.12vs768.67±9.02P<0.01） and its apoptosis rosesmaller too（13.74±1.51vs23.23±1.08P<0.01）.3、Compared to DDP, after the intervention of the combination of NAC andDDP, the concentration of intracellular ROS in A549cells and A549cell spheressignificantly decreased（A549cells547.67±9.07vs768.67±9.02P<0.01，A549cellspheres452.33±16.16vs585.00±12.12P<0.01），and their apoptosis reduced too（A549cells12.47±1.50vs23.23±1.08P<0.01，A549cell spheres8.43±1.10vs13.74±1.51P<0.01）. 4、After the intervention of the combination of GST-π inhibitor BSO and DDP,the levels of GST-π protein in A549cell spheres was lower than A549cells（0.51±0.05vs0.83±0.05P<0.05）, the concentration of intracellular ROS in A549cell spheres was significantly accumulated （1651.00±28.62vs1486.67±10.02P<0.01）, and its apoptosis increased substantially （34.20±2.01vs29.05±1.08P<0.01）.Conclusions:1、The high expression of GST-π protein and low concentration of intracellularROS in A549cell spheres may be one of the reasons for LCSCs resistance tochemotherapy.2、 Antioxidant NAC can effectively eliminate intracellular ROS of cancer,inhibiting the effect of DDP apoptosis.3、BSO inhibits GST-π expression and trigger a large accumulation of ROS, andmediated oxidative stress promotes the chemotherapy apoptosis of A549cell spheres,which can play a sensitizing effect role in chemotherapy.