Study On The Effect And Mechanism Of Autophagy In Sulforaphane-N-acetyl-cysteine Anti-human Glioblastoma Cells

Cerebral glioma is the most common malignant primary neuronal tumor,accounting for 81% of malignant brain tumors,and it is a destructive intracranial tumor and is notorious for its high degree of invasiveness.Gliomas can be graded as I–IV based on malignant behavior by World Health Organization(WHO).Glioblastoma multiform(GBM)is one of the WHO IV grade gliomas and is the most common and fatal adult brain tumor characterized by high growth rate.The tumor is located beneath the cortex with infiltrating growth,often invades several brain lobes and invades deep brain structures.Due to difficulty to be separated and removed completely in a tumor surgery,it often leads to poor prognosis of patients.To date,chemotherapy has become a major treatment modality before or after debulking surgery.However,GBM often exhibits chemotherapy resistance which decreases the patient’s survival rate and shortens the patient’s median survival time.It is urgent to find a new efficient anti-tumor small molecule with low toxicity to normal cells capability to inhibit growth of glioblastoma cells.Since cancer is a group of related diseases,which is characterized by the unlimited growth of tumor cells.Microtubules(MTs),which play a key role in the cell cycle,are "shining stars" for cancer therapeutic targets.Microtubules are basic components of the cytoskeleton of eukaryotic cells.They are hollow tubes composed of 13 aligned protofilaments formed from repeating α-and β-tubulin heterodimers,and play many roles in the cell life activities,such as cell cycle,intracellular protein transport,maintenance of cell morphology,cell division and cell metastasis.α-tubulin,one of heterodimers,is encoded by at least 15 subtype genes in the genome sequencefrom humans,such as TUBA1 A,TUBA1B,TUBA1 C,TUBA3D,TUBA3 E,TUBA4A,TUBA8.Different types of these genes were expressed at different levels and played different roles in a variety of tissues and life processes.Interestingly,latest study showed that increased TUBA1 B expression indicates poor prognosis in liver cancer cells.Epidemiological evidence has been found that agents come from vegetables,sulforaphane(SFN)and its analogs,which have great potential to prevent growth of a variety of tumor cells-lung cancer cells,skin cancer cells,colon cancer cells,breast cancer cells,ovarian cancer cells,for instance.Pharmacokinetic studies in both humans and animals showed that it is metabolized via the mercapturic acid pathway,producing predominantly cysteinylglycine(SFN-CG),cysteine(SFN-Cys),and N-acetyl-cysteine conjugates(SFN-NAC)which are excreted in the urine.SFN metabolites rather than SFN are the major forms of factors in circulation systems and tissues.SFN-N-acetylcysteine(SFN-NAC),one of SFN analogs,was detected in CNS,and it has longer half-time and a higher plasma concentration than SFN with inhibiting histone deacetylase(HDAC)activity.Because of its lipophilicity and molecular size,it is more likely to cross the blood-brain barrier(BBB).At the same time,the presence of SFN could be prolonged by SFN-NAC,it is unstable under physiological conditions and readily dissociated back to SFN.The occurrence of SFN is related to the formation of abnormal mitotic spindle.SFN concentration is higher than the minimum effective inhibitory concentration,and microtubules are solved in human endothelial,mammary gland adenocarcinoma and lung cancer cells.Recently,SFN and its analogs have been shown to combine covalent and apparent selective MTs in A549 lung tumor cells.More and more results showed that SFN and its analogs disrupted MTs in many cancer cells.Autophagy is the general intracellular self-defense system,which includes three types-macroautophagy(hereafter called autophagy),microautophagy,and chaperone-mediated autophagy-and the term“autophagy” usually indicates macroautophagy unless specified which ischaracterized by the formation of double-membraned autophagic vesicles for dysfunctional cellular components,such as misfolded proteins,long-lived proteins,and damaged organelles are sequestered and transported to lysosomes to degradation.In most cases,autophagy is an important mechanism for maintaining cellular homeostasis by nutrition recycling and protein quality control.More evidence showed that autophagy may be activated during chemotherapies in most tumor cell lines.Recent studies show that autophagy play a central role in the pharmacologic manipulation in which its pathways as a therapeutic strategy for cancer.However,whether or not SFN-NAC induced autophagy in human GBM cells and the underlying mechanisms was not clear.Sulforaphane-N-acetyl-cysteine(SFN-NAC)is a metabolite of SFN that has a higher drug concentration and a longer half-life and could cross the blood-brain barrier.Previous studies have found that SFN-NAC can be regulated by ERK to destroy microtubules in lung cancer cells and inhibit cell growth.However,whether SFN-NAC can inhibit the growth of glioblastoma and its related mechanisms are unclear.Here,we demonstrated for the first time that SFN-NAC activates autophagy down-regulation of α-tubulin expression via the ERK pathway.We screened the glioblastoma tumor gene TUBA1 B and predicted its function.U87 MG and U373 MG cells were treated with variouty of concentrations of SFN-NAC.Cell cycle experiments revealed that SFN-NAC induced cell cycle arrested in G2/M phase.Western blot results showed that intracellular ERK activation,autophagy and alpha-tubulin down-regulation were dose-dependent.This condition was reversed using PD98059,an ERK pathway inhibitor.GBM cells were transfected with LC3 siRNA and results showed that inhibition of autophagy reversed downretulion of α-tubulin by SFN-NAC.Further,Co-ip experiments and confocal experiments confirmed that SFN-NAC promotes the binding of LC3 and α-tubulin in the cytoplasm.Cell viability experiments demonstrate that SFN inhibits tumor cell growth.These finding showed that SFN-NAC is a potentially anti-glioblastoma drug.We used Transwell to test the ability ofSFN-NAC to inhibit tumor invasion and inhibit autophagy with 3-MA,a selective inhibitor of autophagy.We found that GBM cells infiltrate after combined use of SFN-NAC and 3-MA.The ability to use SFN-NAC alone is even more pronounced.Subsequently,we examined the expression of cell infiltration-associated protein S100A4,confirming that autophagy induced by SFN-NAC promoted the expression of S100A4.Similarly,the level of apoptosis in SFN-NAC-induced cells was also markedly higher after 3-MA inhibition of autophagy than SFN-NAC alone.These results indicate that in GBM cells,autophagy induced by SFN-NAC promotes cell invasion and antagonizes apoptosis.In this study,taking advantage of Chinese Glioma Genome Atlas(CGGA)data set and TCGA network(http://cancergenome.nih.gov),we gathered samples to take an integrative investigation of α-tubulin gene TUBA1 B and investigated the effect of SFN-NAC on α-tubulin in U87 MG and U373 MG cells,and the underlying mechanisms involved.In summary,we screened the glioma tumor gene TUBA1 B from the glioma patient database,and elucidated the specific mechanism of SFN-NAC down-regulating the α-tubulin protein in GBM cells and the role of autophagy in cell invasion and apoptosis.This study,as a complementary mechanism for SFN-NAC anti-GBM tumors,provides theoretical support for the future clinical application of SFN-NAC.Part one Sulforaphane-N-Acetyl-Cysteine downrugulated α-tubulin via autophagy in U87 MG and U373 MG cellsObjective: Glioblastoma(GBM)is a common primary brain tumor with a very poor prognosis.The standard treatment is surgical resection combined with chemotherapy or radiation therapy.Chemotherapy is its main treatment,GBM is often resistant to the commonly used chemotherapy drug temozolomide(TMZ),making the survival of patients after treatment very short,less than 2 years.Therefore,it is particularly urgent to find a highly effective and safe chemotherapy drug.Epidemiological investigations havefound that ingesting cruciferous plants can effectively reduce the incidence of cancer,and Sulforaphane(SFN)is a natural extract.The results showed that SFN inhibited the invasion of glioblastoma cells through continuous activation of ERK1/2.Sulforaphane-N-acetyl-L-cysteine(SFN-NAC)is an SFN derivative and also SFN.Metabolites in the human body,with longer half-life and better ability to cross the blood-brain barrier(BBB),may be more effective in inhibiting tumor growth.This experiment will study the mechanism of SFN-NAC inhibiting the growth of glioblastoma U373 MG and U87 MG cells and provide theoretical support for clinical treatment of glioblastoma patients.Methods: The ability of SFN-NAC to inhibit GBM cell growth was detected by using cell proliferation assay.Morphological changes of the cells were observed by microscope.α-tubulin,LC3II/LCI ratio and phosphorylation of ERK were detected by Western blot.The co-immunoprecipitation assay(Co-IP)and electronimmune confocal microscopy(Confocal)were used to detect the binding ability of LC3 toα-tubulin.LC3 siRNA was used to knock down the expression of LC3.Transmission electron microscopy(TEM)was used to observe the cellular ultrastructures,such as autophagosomes and autolysosomes.Results: Morphology was markly changed When U87 MG and U373 MG cells treated with SFN-NAC on 30 μmol/L,SFN-NAC inhibited the viability of U373 MG and U87 MG cells in a dose-dependent manner;cell viability also began to decrease significantly,30 μmol/L is the optional concentration;Western blot shows that SFN-NAC down-regulates alpha-tubulin,up-regulates LC3II/LCI ratio and activates ERK phosphorylation in a dose-dependent manner;these results are reversed after using the ERK pathway inhibitor(PD98059)After transfection of cells with LC3 siRNA,the down regulation ofα-tubulin was reversed;Co-Ip and confocal results showed that SFN-NAC enhanced the binding ability of LC3 II to α-tubulin.It was shown that SFN-NAC increases the formation of autophagosomes and autolysosomes.Part two Autophagy induced by Sulforaphane-N-Acetyl-Cysteine promotedinvasion and inhibited apoptosis in U87 MG and U373MG cellsObjective: Glioblastoma is the brain tumor with the highest degree of malignancy.It has extremely high invasive ability and extremely high growth rate.Controlling cell invasion and inducing apoptosis is an important strategy for the treatment of tumors.Autophagy as a highly conservative self-degradation metabolic mechanism of the cell itself plays an important role in the treatment of tumors.Studies show that autophagy can promote tumor resistance,antagonize apoptosis,and promote tumor cell survival in tumor therapy.Infiltration is promoted by cell epithelial-mesenchymal transition;on the other hand,excessive autophagy can lead to autophagic apoptosis,also known as early stage II programmed death results,SFN and some of its derivatives can inhibit glioblastoma cells invasion,promote apoptosis.This study will investigate the relationship between autophagy and tumor invasion and apoptosis in glioblastoma U373 MG U87MG and cells treated with SFN-NAC,providing theoretical support for clinical treatment of glioblastoma patients.Methods: Invasion assay(Transwell)was used to detect the inhibitory effect of SFN-NAC on the invasion of glioma cells.The ability of SFN-NAC to induce apoptosis was detected by flow cytometry.Western blot was used to detect the expression of intracellular protein s100A4,and autophagy inhibitor3-MA was used to inhibit tumor cell autophagy.Transmission electron microscopy(TEM)was used to observe the ultrastructure of cells such as apoptotic bodies in cells.Results: SFN-NAC induces apoptosis of glioblastoma and follows a dose-dependent relationship.Apoptosis experiments showed that when the concentration of the drug was 30 μmol/L,the addition of self-selective inhibitor(3-MA)promoted apoptosis.Transwell experiments showed that SFN-NAC concentration was 10μmol/L,and the combination of 3-MA inhibited the invasion of cells.Western blot results showed that SFN-NAC30(μmol/L)combined with 3-MA reduced s100A4 than single use.The SFN-NAC reduced s100A4 more significantly.Part three Expression and functional analysis of TUBA1 B Gene in glioma patientsObjective: There are 15 subtypes of α-tubulin in the human body,and are encoded by different mRNAs.They are TUBA1 A,TUBA1B,TUBA1 C,TUBA3D,TUBA3 E,TUBA4A,TUBA8,etc.TUBA1 B is one of them and highly expressed in human glioma cells.In other cancers,it has been found to be highly correlated with clinical information such as tumor grading and prognosis.To clarify the expression and clinical significance of TUBA1 B in gliomas,we analyze and predict its function used in glioma patients data.Methods: T-test,Kaplan-Meier survival analysis and Cox survival analysis were used to evaluate the relationship between TUBA1 B gene and clinical information in patients with glioma.Using G.O analysis to predict the function of the TUBA1 B gene,P<0.05 was considered statistically significant for all statistical methods.Results: The expression of TUBA1 B increased with the increase of glioma grade.In the survival analysis,patients with high expression of TUBA1 B had lower survival periods than patients with low expression of TUBA1 B.The age,tumor grade,and IDH mutation type of TUBA1 B patients were independent risk factors for the prognosis of patients.TUBA1 B is closely related to cell cycle.Conclusions:1.SFN-NAC as a natural food source molecule with a low toxicity and good efficiency to anti-tumor cells.It inhibits cell growth via destroying cell microtubule structure in GBM cells.2.SFN-NAC regulates autophagy through activation of ERK1/2 in glioblastoma cells and in turn down-regulates α-tubulin expression.This is an important mechanism for the treatment of glioma by α-tubulin protein compiled by SFN-NAC targeting glioma cancer gene TUBA1 B.3.Autophagy induced by SFN-NAC promotes cell invasion and antagonizes apoptosis.