Detection Of Selenoelen And Reactive Oxygen Species In The Process Of Apoptosis Of Tumor Cells Induced By Na ₂ SeO ₃

Selenium is an essential trace element, and is closely associated with many human diseases.Sodium selenite as an anti-cancer drug, can be effective in killing tumor cells, with less toxic tonormal cells. With the increase in the incidence of cancer, the anti-cancer effect of sodiumselenite causes more attention. However the specific mechanism of anti-cancer of sodiumselenite is still unclear. A large number of studies suggest that selenols generated in themetabolic process of sodium selenite can react with oxygen to produce reactive oxygen species,causing oxidative stress and leading further to tumor cell death. However, in the previous study,the hypoxic microenvironment of solid tumors was ignored and most of researches was focusedon oxidative stress associated with various signaling pathways in the normoxic environment.Therefore, we designed and synthesized two fluorescent probes for selenols, and observed theconcentration changes of selenols and reactive oxygen species in the sodium selenite-inducedapoptotic process of tumor cells. And we tried to explain the mechanism of sodium seleniteanti-cancer in the real microenvironment by observing the redox state.This paper reviews the research status and development trend of selenols and simultaneousdetection of selenols and H2O2, with the selenite-induced apoptosis of hepatoma cell model.Two novel nanoprobes for selenols were designed with gold nanoparticles and peptide chainand the simultaneous detection of selenol and reactive oxygen species were completed in livingcells. The specific content of the work is divided into the following three aspects:Firstly, we designed a new near-infrared (NIR) gold nanoprobe for imaging selenols inliving cells, based on an assembly of the modified peptide chains onto the surface of goldnanoparticles (AuNPs) via Au-S bond. The peptide chain is modified with the near-infrared dye(N terminal Cy5.5, C-terminal cysteine exposed mercapto). Fluorescence resonance energytransfer (FRET) occurs between the Cy5.5dye and gold nanoparticles, and the fluorescence ofdye is quenched. When selenol is present, the connection (Au-S) of the peptide chain includingCy5.5dye and AuNPs is replaced with Au-Se, resulting in the fluorescence recovery of Cy5.5dye, because the nucleophilicity of selenol (-SeH) is much greater than that of thiol (-SH). Thus,a specific method of detecting and imaging selenols is established.Excitation and emission of the probe are in the near infrared region, avoiding the interference of organism’s auto-fluorescence, compared with the previous visible probe. Andthe probe is highly selective and sensitive to selenols, but the responses of the probe towardreducing substances, oxidizing substances, amino acids, metal ions etc were negligible. In therange of concentration from0to100μM, the fluorescence increase is proportional to theconcentration changes of selenol. The regression equation is F=1623.3+170.18[Sec] μM,with a linear correlation coefficient of0.9902. The cytotoxicity assay proved gold nanoprobehas good biocompatibility and low toxicity. Under hypoxic conditions, it can detect selenolcontent in selenite-induced apoptosis of hepatoma cells.Secondly, a visible gold nanoprobe was synthesised for detecting selenol in living cells. Inthe human body, sodium selenite is metabolized to product selenol and reactive oxygen species.Therefore simultaneous detection of selenol and reactive oxygen species under hypoxia iscontributed to the research of sodium selenite’s anti-cancer mechanism. Based on the strategyof same excitation and two different emissions, we designed a wavelength matching goldnanoprobe for detecting selenol. The modified peptide chains (N terminal FAM, C-terminalcysteine exposed mercapto) are assembled onto the surface of AuNPs via Au-S bond. Whenselenol is present, the Au-S bond is replaced with Au-Se bond, resulting in the fluorescencerecovery of FAM dye, and the excitation and emission of are in visible region (Ex=490nm,Em=520nm). Respectively, the probe was analyzed in the chemical and biological modelsystems. The probe had the capable of specifically detecting selenol, without the interferenceof some reducing substances, oxidizing substances, amino acids, metal ions etc. It exhibited ahigh selectivity and sensitivity.In the range of concentration from0to100μM, the fluorescenceincrease is proportional to the concentration changes of selenol. The regression equation is F=2729.79+308.65[Sec] μM with a linear correlation coefficient of0.9959. The Au nanoprobehad a good biocompatibility and low toxicity. Under hypoxic conditions, it could detect thechanges of selenol in the process of selenite-induced apoptosis in tumor cells.Thirdly, under the same excitation, we simultaneous detected selenol and H2O2. On thebasis of the previous chapter, we choose a QCy7probe to the specific detection of H2O2,which has phenylboronic acid attached through an ether-linkage to QCy7. The probe exhibitstwo excitation peaks and a NIR emission peak (Ex1=490nm, Ex2=560nm, Em=710nm).With the last chapter, the excitation spectra of the two probes are overlaped, and the emission spectra are separation. We achieved the same excitation simultaneous detection of selenol andH2O2in the chemical system very well.There is no interference between the two probes. In thecondition of normoxic/hypoxic, selenol and reactive oxygen species are detected in selenite-induced liver cancer cells. The results found that, under normoxic conditions,with the increaseof sodium selenite induced time, selenol and intracellular H2O2are significantly increased.When the lack of oxygen, with the increase of sodium selenite-induced time,intracellularselenol is increased, however the content of H2O2is unchanged. The reason may be that in thehypoxic tumor microenvironment, the content of oxygen is low, it can’t produce more ROSwith selenol. Experiment result showed that the sodium selenite induced apoptosis of tumorcells is not a oxidative stress. It could be a reduction stress caused by high concentration ofintracellular selenol. In this study, we provided a new thought for the anticancer mechanism ofsodium selenite in the future.