Resveratrol And Its Nanoparticles Induced Ferroptosis In Glioblastoma Cells In Vitro And In Vivo Via Improve Resveratrol Bioavailability

Background:Glioma is the most common primary intracranial malignant tumor,accounting for 81%of central nervous system（CNS）malignant tumors.According to the data from World Health Organization（WHO）,gliomas are classified into 4 grades,Grade 4 gliomas are high-grade and extrremely lethal.Generally,higher grade tumors are associated with poor prognosis,and glioblastoma（GBM）is the most common type of Grade 4 glioma,with rapid and agrassive growth.Currently,the clinical countermeasures of GBM are mainly surgical resection,combined by radiotherapy and chemotherapy.Nevertheless,the therapeutic efficacy is unsatisfactory due to the drug resistance and tumor relapse.Consequently,the prognosis of GBM patients remains very poor.Moreover,conventional chemotherapy drugs have strong side effects,resulting in multiple body damages and therefore seriously affect the life quality of GBM patients.It is thus in urgent need to explore anti-GBM drug（s）that can effectively inhibit the GBM growth with little toxic effects.Resveratrol is a non-flavonoid polyphenol organic compound,which is rich in grape leaves and grape skins.It has a wide range of biological effects,including tumor prevention and treatment.Our previous studies found that this compound had strong inhibitory effects on thyroid,bladder and ovarian cancer via inhibiting the activities of cancer-associated signaling pathways such as STAT3,Wnt and Notch.Ferroptosis is a form of iron dependent cell death caused by accumulation of the toxic lipid hydrogen peroxide（ROS）.A body of evidence suggests that glutathione peroxidase 4（GPX4）can be used as an indicator of iron death in cells through degrading small molecule peroxides and some lipid peroxides,and inhibit lipid peroxidation.SIt has been found that GPX4 expression levels are significantly increased in tumor tissues,such as colorectal adenocarcinomas,prostate cancers,and GBMs,compared with their normal counterparts.The highly expressed GPX4 is closely related to the poor prognosis of GBM patients.This findings suggest that cancer cells with high GPX4 expression are more snsensible to the killing effect of the drug by resisting ferroptosis.Drug resistance has been a tough problem in cancer therapy.The discovery of GPX4 provides a potential molecular target to overcome drug resistance.We have found that reactive oxygen specise（ROS）is a possible factor in determining the differential resveratrol sensitivity between U251 and LN428 glioblastoma cells.GPX4 can reduce ROS to the corresponding alcohols,thereby inhibiting the iron death of cells.For these reasons,we consider that GPX4 may be another influent factor for resveratrol sensitivities of glioblastoma cells.In parallel,we prepared targeted nanoparticles loaded with resveratrol（Pep-PP@Res）to improve the in vivo/intracellular bioavailability of resveratrol and its anti-GBM effect,because of the shortcomings of resveratrol,such as short half-life in vivo,low bioavailability in the brain and unsuitable for clinical application.This study aims to address:1)whether ferroptosis is one of the forms of anti-GBM effects of resveratrol;2)whether GPX4 is a determining factor for the differential response of GBM cells to resveratrol;3)the relevance of GPX4 expression with ferroptotic susceptibility;4)the ability of resveratrol targeting nanoparticles Pep-PP@Res to cross the blood-brain barrier,to improve resveratrol intracerebral bioavailability and to target the transplanted tumors.The findings of current study may provide new experimental evidence for the application of resveratrol in the treatment of GBMs in terms of its mechanism of action and practicability.Methods:U251 and RG2 sensitive to resveratrol and LN428 glioma cell lines resistant to resveratrol were selected in this study.The sensitivity of glioma cell lines to resveratrol was determined by CCK-8（Cell Counting Kit-8）method.hematoxylin/eosin（HE）to observe the morphological changes of cells treated with resveratrol.DCFH-DA,C11（C11BODIPY581/591）and PGSK(Phen Green^TM SK dipotassium salt)staining kits to detect intracellular ROS,the content of lipid peroxide and Fe²⁺,determine whether resveratrol caused death of iron.Immunofluorescence,Western blot to study the changes in the expression of GPX4,a key protein of iron death after resveratrol treatment,and to analyze the correlation between the difference of resveratrol sensitivity and iron death in glioblastoma.According to the different GPX4 expression of U251 and LN428 cell lines induced by resveratrol,the GPX4 inducer Liprostatin-1 was added to U251,and the GPX4 inhibitor RSL-3 was added to LN428.HE,DCFH-DA,C11,PGSK and WB tests were used to further explore whether the differences in the sensitivity of U251 and LN428 cell lines to resveratrol were related to GPX4.100?M Resveratrol treatment rat RG-2 cell lines after the observation of cell morphology change,iron death index ROS,the levels of lipid peroxides,Fe²⁺and GPX4 expression.According to the difference in the expression of IL-13Rα2 between RG-2 and normal rat brain tissue,Res and its targeted nanoparticle Pep-PP@Res were used to treat RG-2transplanted rat tumors and detect the expression of GPX4 in the tumor.In order to prove the Pep-PP@Res can through the blood brain barrier,improve the bioavailability,and further investigate whether the Pep-PP@Res in situ tumor has targeted,first in vitro cultivation of RG-2 cells,giving 100μM and200μM respectively of Resveratrol（Res）and nanoparticles（Pep-PP@Res）,gradient time after drug treatment using high performance liquid chromatography High Performance Liquid Chromatography;The absorption of two Res preparations by RG-2 cells was determined by HPLC.Finally,intracranial glioblastoma model of rats was established,and intravenous,nasal and abdominal administration methods were designed to check the bioavailability and targeting distribution of the drugs in the brain.Results:（1）The results of CCK-8 assay and HE staining shows that U251 are more sensitive to resveratrol after 48 hours of resveratrol treatment,and the cells are drawn and wrinkled,growth and proliferation significantly inhibite,and the number of cells significantly decreases.On the contrary,the morphology of LN428 don’t change significantly after 48h of resveratrol treatment,and the cell proliferation is not inhibited.（2）Experimental results of DCFH-DA,C11 and PGSK demonstrate that:After 48h of resveratrol treatment,ROS,lipid peroxides and Fe²⁺increase significantly in U251 cells,with the range of 51.5%,105.4%and 42.6%,respectively,suggesting that resveratrol can induce ferroptosis in U251 cells,while the above indexes do not change significantly in LN428 cells treated with resveratrol.（3）GPX4 are highly expressed in U251 and LN428 cells during normal culture,IF and Western Blot detection show.GPX4 expression in resveratrol treated U251 cells decreases（1.32 and 1.53 fold）,but there is no difference in LN428 cells.（4）After adding Lipro-1 and RSL-3,the staining results of HE,IF and Western Blot showed that:Compared with resveratrol alone,U251 cells treated with Lipro-1 combined with resveratrol has a higher number of cells,a slower change in cell morphology,and a 98.9%and 38.7%increase in GPX4 protein level compared with resveratrol alone.LN428 cells treated with RSL-3 combined with resveratrol shows more significant shrinkage,number reduction and downregulation of GPX4 expression,which decrease by 35.4%and 65.8%compared with those treat with resveratrol alone.（5）RG-2 cell line is sensitive to resveratrol,and resveratrol down-regulates GPX4 expression;（6）HPLC results show that Pep-PP@Res can significantly improve the uptake capacity of granules and prolong the residence time of resveratrol in cells in vitro,thus enhancing the antitumor effect of resveratrol.In vivo experiments show that Pep-PP@Res can pass the blood-brain barrier,improve bioavailability and enrich drugs at the tumor site.（7）IL-13Rα2 is overexpressed in glioblastoma and under expressed in normal brain tissue,and GPX4 expression is significantly down-regulated after Pep-PP@Res treatment,which effectively inhibits the growth of orthotopic grafts by inducing ferroptosis.Conclusions:（1）Resveratrol induced the ferroptosis of U251 cells that are sensitive to it but not LN428 cells resistant to it;（2）Resveratrol down-regulates GPX4 expression in U251 cells;（3）RG-2 cells are sensitive to resveratrol and show GPX4 down-regulation upon resveratrol treatment;（4）Pep-PP@Res significantly improves bioavailability of GBM cells in vitro and in vivo,especially in rat RG2 orthotopic transplanted tumors.（5）In vivo experiments confirm that resveratrol and its targeted nanoparticles Pep-PP@Res can induce ferroptosis in GBM cells and significantly down-regulate GPX4 expression.This study demonstrates that GPX4downregulation and ferroptotis induction is the novel cellular and molecular biological parameters of resveratrol efficacy against glioblastomas.