A Colorimetric And Ratiometric Fluorescent Probe For Detection Of Glutathione During Oxidative Stress And Apoptosis Of Oral Squamous Cell Carcinoma Cells

Objective:Oral squamous cell carcinoma(OSCC)is the most common malignant tumor in head and neck region.Although the comprehensive and sequential treatment have been applied in treatment of OSCC,but there are still high recurrence rate and metastasis rate.Especially for patients with advanced state,survival rate of 5 year is still quite low,and the prognosis is poor.Chemotherapy is one of the main treatment methods for oral squamous cell carcinoma,but the emergence of more and more chemotherapy drug resistance is the biggest bottleneck which restricts the effect of chemotherapy.One of the most important mechanism of some commonly used chemotherapeutic drugs,including cisplatin,is to induce formation of reactive oxygen species(ROS).At the same time,in mammalian cells,there are a lot of enzymatic or non enzymatic antioxidants to be against oxidants including ROS.Under physiological conditions,intracellular oxidants and antioxidants maintain a state of dynamic equilibrium.On the other hand,antioxidants may also counteract ROS and weaken or suppress the role of chemotherapy agents.Glutathione(GSH),as the main redox regulator,tightly controls the intracellular redox balance,and also closely related with drug resistance.To date,a number of specific GSH fluorescent probes have been developed that can selectively and quantitatively detect alteration of intracellular glutathione.However,as far as we know,there is not any kind of probe to be used to show the process of promoting apoptosis by inducing modification of celluar redox state.This time we applied a novel GSH selective fluorescent probe to detect alteration of GSH level of oral squamous carcinoma cells(OSCCs)during the course of oxidative stress and apoptosis induced by exogenous oxidative damage and depletion of mitochondrial GSH.Through this study,we wanted to further study the effect of GSH on cellular oxidative stress and apoptosis,especially in mitochondria.Methods:1.Application of the GSH probe to assess GSH levelsAfter oral squamous cell carcinoma cells Cal-27 were treated with 100μM H2O2,1Mm NAC and 50μM EA consecutively,the GSH probe is used to demonstrate the alteration of GSH contents.Under confocal microscopy blue fluorescence represents intracellular GSH.2.Assessment of intracellular ROS and GSH levelsAfter oral squamous cell carcinoma cells Cal-27 were treated with 100μM H2O2 and 50μM EA consecutively,The levels of GSH and ROS were detected by GSH probe and DCF-DA probe.Under the confocal microscope,the blue fluorescence represents the intracellular GSH level,and the green fluorescence represents the level of intracellular ROS.3.Assessment of mitochondrial ROS and GSH levelsAfter oral squamous cell carcinoma cells Cal-27 were treated with 100μM H2O2 and 50μM EA consecutively,we applied the Red CM-H2XRos Mitotracker probes to locate the mitochondria,the GSH probes and the DCF-DA probes to detect changes in the levels of GSH and ROS in mitochondria.Under the confocal microscope,the red fluorescence represents the mitochondria,the blue fluorescence represents the intracellular GSH concentration,and the green fluorescence represents ROS concentration.4.Annexin V-FITC staining for apoptosis and assessment of GSH levelsAfter oral squamous cell carcinoma cells Cal-27 were treated with 100μM H2O2 and 50μM EA consecutively,apoptosis was detected by V-FITC Annexin probe and the GSH probe was used to detect the alteration of cellular GSH contents.Under the confocal microscope,the green fluorescence represents the occurrence of apoptosis,the blue fluorescence represents intracellular GSH concentration.5.Assessment of mitochondrial membrane potential(△φm)and GSH levelsAfter oral squamous cell carcinoma cells Cal-27 were treated with 100μM H2O2 and 50μM EA consecutively,the JC-1 probe was used to detect the mitochondrial transmembrane potential,and the GSH probe was used to detect the alteration of GSH concentration in the cells.Under the confocal microscope,the red fluorescence represents the occurrence of mitochondrial transmembrane potential,and the blue fluorescence represents intracellular GSH concentration.Results:1.Before treatment the cells showed intensive blue fluorescence,after 100μM H2O2 was added to the cells for 30 min,decrease of the blue fluorescence could be observed.Then the cells were treated with ImM NAC for 1h,and the cal-27 cells emitted significant blue fluorescence again.Finally after 50μM EA was added to the cells for another 30 min,the blue fluorescence declined obviously to almost disappearance.2.Before treatment the cal-27 cells displayed intensive blue fluorescence and faint green fluorescence,which indicated high levels of GSH and low levels of ROS.After the cells were treated with H2O2,green fluorescence was enhanced and blue fluorescence was weakened,which indicated ROS levels increased and GSH levels decreased.Especially after the cells were treated with EA,we could see that green fluorescence increased and blue fluorescence decreased more obviously compared with the effect of H2O2.3.Before treatment the cal-27 cells displayed intensive blue fluorescence and faint green fluorescence described as above.And the merge of mitochondria and GSH displayed intense purple fluorescence which indicated high mitochondrial GSH levels,the merge image of mitochondria and ROS displayed feeble orange fluorescence which indicated low mitochondrial ROS levels.After the cells were treated with 100pM H202 for 30 min,the blue fluorescence attenuated and the green fluorescence intensified.Accordingly merged purple fluorescence attenuated and merged orange fluorescence intensified which indicated that mitochondrial GSH levels decreased and mitochondrial ROS levels increased.Then after 50μM EA were added to the cells for the next 30 min,a significant decrease in blue emission and a significant increase in green emission could be viewed.And the merged purple fluorescence attenuated and the merged orange fluorescence intensified obviously4.Before treatment the Annexin V-FITC probe in the cal-27 cells displayed feeble green fluorescent and the GSH probe emitted intense blue fluorescence.Along with the cells were added with 100μM H2O2 for 30 min,blue fluorescence attenuated and green fluorescence intensified.Subsequently after the cal-27 cells were treated with 50μM EA for the next 30 min,blue fluorescence nearly disappeared and the cells give rise to a significant increase in green emission.5.Before treatment,high levels of GSH in the cal-27 cells emitted intense blue fluorescence.After the cal-27 cells were treated with H202 and EA consecutively,blue fluorescence weakened rapidly which indicated intracellular GSH levels decreased.At the same time,loss of mitochondrial membrane potential led to decomposition of the JC-1 polymer and a significant decrease of red emission in the cal-27 cells.Conclusions:In summary,our study showed that the GSH selective fluorescent probe can be applied to detect alteration of cellular GSH levels sensitively during the course of oxidative stress and apoptosis of OSCCs induced by exogenous H2O2 and enhanced by depletion of mtGSH.The greatest advantage of the GSH probe is that we can visualize the intact process in real time and quantitatively.At last we can draw the conclusion that depletion of mtGSH could enhance apoptosis of the cal-27 cells induced by oxidative stress.