Effect Of Intracellular ROS Levels Change On Cell Activity And Related Signal Transduction Pathways

Cell injury and carcinogenesis from oxidative stress have been implicated in diverse trauma and tumor disease. Reactive oxygen species (ROS) are constantly generated and eliminated in the biological system, and expression levels of ROS are different in different cells. More importantly, basal ROS expression levels are much lower in normal cells, compared with the tumor counterparts. ROS play important roles in a variety of physiological and pathological processes, and ROS may also function as secondary messengers in pathways, such as the Akt pathway, mitochondria-mediated apoptosis pathway,and regulation of cell cycle proteins to inhibit the cell proliferation pathway. ROS must be strictly controlled in the biological metabolic processes, attributed to its complex role. Any change in the level of intracellular ROS is likely to lead to changes in cell activity. However, the complete signaling pathways involved are still unclear, this study will construct intracellular high-ROS environment or low-ROS environment, study of effect on cell activity will be performed, and then a more complete signal pathway will be established, which provide the experimental basis to elucidate the complex role of ROS in the cells, and theoretical support for the mechanism of action of ROS-related drugs.The main results of this study are as follows:1. We describe the properties and biological applications of three fluorescent probes for detecting H2O2in living tumor cells. Comparing the three popular probes, based on their sensitivity the kinetics of H2O2fluctuation and cellular distribution, our study illustrates that as a fluorescent probe, dihydrorhodamine123is more appropriate probe for intracellular H2O2investigations.2. We selected three tumor cell lines as subjects, H2O2or NAC has emerged as a phenomenon of inhibition of tumor cell activity in a distinct dose-and time-dependent manner. H2O2more effectively inhibits SPC-A-1lung adenocarcinoma cells proliferation; meanwhile, HepG2the human liver carcinoma cells are more susceptible to NAC-induced anti-proliferation. The analysis of the intracellular redox state showed that:H2O2effectively increased the oxidative stress in the three kinds of tumor cells, NAC showed the opposite result, which confirmed again that the change of intracellular redox state may affect cell activity. It is worth noting:the H2O2-induced intracellular redox state change is more obvious in the SPC-A-1cells and NAC induced intracellular redox state change is more pronounced in HepG2cells, which is consistent with the results of the cell activity. These results also suggest that change of intracellular ROS level would influence cell fate.3. We further study the mechanism of H2O2-reduced tumor cell activity, and clarify the high oxidative stress-induced intracellular signaling pathway. The results clearly demonstrate that:increased oxidative stress modulates explosive efflux of cytosolic Ca2+, and concomitant mitochondrial Ca2+uptake, further induce ROS amplification, and in turn high ROS levels facilitate Ca2+accumulation by release from internal stores and impairment of Ca2+clearance systems. Changes of apoptotic proteins and anti-apoptotic protein expression level lead to cyt c release from mitochondria, and activate related casepase activity, ultimately leading to cell apoptosis.4. we have studied the mechanism of antitumor activity of the two substances (Tα1,FLZ), their common feature is the ability to reduce the level of intracellular oxidative stress. Tal can effectively reduce ROS levels and increase intracellular GSH levels in HepG2human hepatoma cells, suppressor cells in the G1phase, thereby reducing cell activity; however, Tal showed the opposite results in mice spleen leukomonocytes. FLZ can effectively inhibit the activity of tumor cells, but has little toxicity to normal liver cells, which may be associated with lower background level of ROS in normal cells. Our research suggest that FLZ has potent anti-proliferative activity against malignant human hepatoma cells via modulation of the expression or activation of cell-cycle regulatory proteins, which are associated with decreased Ca2+/ROS levels. In short, if a material can effectively reduce the level of ROS in tumor cells, it has a certain anti-tumor activity in theory, but as a potential treatment is the need for further research. 5. Finally, we explored the role of the ASPP family in the high oxidative stress-induced apoptosis, overexpression of iASPP protein by cell transfection can inhibit apoptosis induced by H2O2, which may reveal the link between the ASPP family and ROS, and provide new ideas to further improve the signal transduction pathway of ROS-induced apoptosis.In summary, our studies confirm that changes in the level of intracellular ROS can affect cell activity, and explain the difference of drug sensitivity in tumor cells and normal cells; meanwhile, we first reveal the important role of the ASPP family in the high oxidative stress-induced apoptosis. These findings will contribute to develop ROS-related anticancer drugs.