ROS Levels In Resveratrol-treated Glioblastoma Cells And Their Relevance With Chemosensitivities

Backgrounds: Glioma is a major type of malignant brain tumor in adults.Glioblastoma(GBM)is the most malignant brain tumor accounts for 45.6% of all primary malignant brain tumors,which is the most common and difficult to cure.Currently,surgical resection is the preferred method of glioblastoma,however,due to its highly aggressive and diffuse growth,simple surgery is difficult to completely remove it,patients always have poor prognosis after surgery and the condition is easy to repeat.Therefore,postoperative radiotherapy and chemotherapy has become a positive adjuvant therapy.Studies have shown that surgery combined with radiotherapy and chemotherapy treatment can effectively extend the survival time of patients.Even though,5-year survival rate of patients is still below 10%.At the same time,conventional glioma chemotherapy drugs have toxic side effects,can easily cause the central nervous system and even the entire body damage,and due to the presence of blood brain barrier(BBB),some drugs are hard to pass,chemotherapy effect is difficult to achieve the desired level.Therefore,the search for an anti-glioma drug that can effectively inhibit the growth of tumor cells,limit the invasion of tumor cells and non-toxic side effects is of great clinical significance.Resveratrol,3,5,4'-trihydroxystilbene,is a polyphenolic compounds derived from grapes,mulberry and other plants.It has a wide range of health benefits including anti-inflammatory,anti-tumor and prevent cardiovascular disease.This nontoxic compound not only possesses cancer preventive properties but also exerts anticancer effects on many types of cancers.Reactive oxygen species(ROS),mainly including highly active ions and molecules,are generated as the mitochondrial by-products of oxygen metabolism.Under normal circumstances,the body of reactive oxygen species from the body to maintain a relatively balanced antioxidant status.Mainly including antioxidant enzymes such as superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GPx)and some small molecule substances Such as vitamin A,C and reduced glutathione(Reduced Glutathione,GSH)and so on.At present,studies have confirmed that ROS can act as a signaling molecule to mediate cell signal transduction and act on mitochondria to induce apoptosis.Therefore,through the use of drugs to break the balance of redox balance within the tumor cells,increase the level of intracellular ROS expression,leading to oxidative stress and induce apoptosis of cells,may serve as another approach to cancer treatment.Antioxidant activity is known as one of the beneficial effects of resveratrol on normal cells,while the corresponding data from cancer cells remain lesser known.Recently,we found abundant spheroid mitochondria,an indicator of oxidative mitochondrial damage,in resveratrol-sensitive ovarian cancer cells.This phenomenon suggests that resveratrol may increase rather than reduce oxidative stress in cancer cells presumably due to the poorly operated intracellular resveratrol metabolic machinery in cancer cells.In view of the above,we think that the oxidative status may be a possible factor to determine the resveratrol sensitivity of glioblastoma cells.To this end,the main purpose of this experiment: 1.To explore the sensitivity of glioblastoma cells U251,LN428 to resveratrol.2.Electron microscope observation of glioblastoma cells U251,LN428 after resveratrol mitochondria whether the abnormal morphology.3.To detect the expression of ROS in U251 and LN428 cells treated with resveratrol and the expression of related antioxidant enzymes.4.Explore the mitochondria-mediated apoptosis pathway is closely related to caspase protein expression in glioblastoma U251,LN428 before and after treatment.5.Detect the expression levels of resveratrol-related metabolic enzymes SULT 1A1 and 1C2 before and after treatment with glioblastoma U251 and LN428.Method: Using multiple experimental approaches tests Resveratrol sensitivities of two GBM cell lines(U251 and LN428).The statuses of ROS expression are evaluated by Flow cytometry and fluorescent microscopic.Immunohistochemical staining and Western bolt are conducted to profile SOD2,CAT,caspase-9 and-3 and SULTs expression patterns in two GBM cells.Result: 1.U251 glioma cells treated with resveratrol significantly change the cell morphology,cell proliferation was significantly inhibited,the number of cells was significantly reduced,TUNEL positive expression was significantly.The cell morphology of LN428 glioma cells treated with resveratrol basically did not change,the number of cells was not significantly inhibited,TUNEL expression was negative.2.U251 cells treated with resveratrol displayed typical apoptotic morphological changes such as chromatin condensation and fragmentation.In comparison with the intact mitochondria of the control cells,the double membrane defined mitochondrial spheroids were commonly found in resveratrol-treated U251 cells.In contrast,the ultrastructures of 48 hours resveratrol treated LN428 cells remained identical with that of the control cells and no mitochondrial spheroid was found in them.3.Fluorescence staining results of DCFH-DA probe cells and flow cytometry were quantitatively found.After resveratrol treated U251,the green fluorescent intensity increased at 24 h,but the fluorescence intensity of LN428 cells did not occur Variety.The result of ICC and Western Blotting showed that the level of SOD2 and CAT in resveratroltreated U251 cells decreased and showed a time-dependent manner,while the level of SOD2 and CAT in LN428 cells was not significantly decreased,indicating that resveratrol did not.4.Western Blotting was used to detect the expression of pro-caspase-9 and-3,activecaspase-9 and-3 in U251 and LN428 cells.After treated with resveratrol for 48 h,the levels of pro-caspase-9 and-3 in U251 cells decreased slightly,while the active caspase-9 and-3 were significantly up-regulated.The above parameter level of LN428 cells remained almost unchanged,not related to resveratrol treatment.5.The relative expression of SULT 1A1 and 1C2 in U251 glioma cell line before treatment with resveratrol was weaker.After resveratrol treatment,the expression of SULT1A1 and SULT1C2 increased slightly,but the expression level of SULT1A1 and SULT1C2 was lower than that of LN428 Tumor cell line.The expression of SULT1A1 and SULT1C2 in LN428 glioma cell lines was relatively strong.After resveratrol treatment,the expression of SULT1A1 and SULT1C2 increased to a certain extent.Conclusion: 1.Resveratrol can increase the production of ROS in U251 cells,which induce oxidative stress and induce apoptosis.2.Resveratrol down-regulated the expression of SOD2 and CAT in its sensitive cell line U251.3.The mechanism of resveratrol-induced GBM apoptosis may be through the increase of ROS production,resulting in mitochondrial oxidative damage,eventually leading to the activation of caspase-9 and caspase-3.4.The reason of resveratrol sensitivity may be negatively correlated with the expression level of its metabolic enzyme SULTs,that is,the higher the expression of metabolic enzymes,the worse the sensitivity of resveratrol.