| Background and purposeMucin-type O-glycosylation is the most pervasive form of protein glycosylation. Its initial response was catalysed by peptide: N-acetyl-galactosyltransferase. Human UDP-GalNAc: Polypeptide N-Acetylgalactosaminyltransferase is a glycosyl- transferase involved in the first step of O-glycan synthesis. It catalyses the transference of GalNAc group from UDP-GalNAc to the threonine (Thr)or serine (Ser) residue within a definite sequence on protein polypeptide chain to form a GalNAcα-O Thr/Ser glycosidic linkage. Studies have shown that O-sugar chain play an important role to many cell functions, such as the growth and development et al. Suppression and removal of cell surface sugar chains will result in cell growth and behavior change, and even apoptosis, death. Previous research revealed that ppGalNAcT maybe concerned with the growth and transfusion of tumor. PpGalNAc-T2 has a widely distribution and substrates spectrum in its family, so it is perhaps the rate-limiting enzyme of O-glycosylation, have important biological significance so this article chose it to study.Leukemia is a kind of clonal malignant disease of hematopoietic stem cells. Leukemia in cancer mortality is at the top of the list under 35 years of age, and its incidence has increased year by year. Although in the past few decades the treatment of leukemia has made significant progress, treatment-related toxic side-effects and drug resistance seriously impact patient's overall survival. Arsenic trioxide was found in Chinese Medicine, it can be used for the treatment of acute promyelocytic leukemia. Vanadium is a trace element known to be essential for a number of species that is widely distributed on earth. During the last few decades, the facade of vanadium as a "slightly" toxic and carcinogenic element eventually was acknowledged to be an essential trace element with anti-diabetic and anti-carcinogenic properties. In recent years, researchers have continued to deepen understand the chemical mechanism of vanadium. Researchers found that vanadium compounds can be used as a potential therapeutic agent. At present, design and find effective low toxicity drug to treat leukemia has become a research direction. Whether Arsenic trioxide combined with sodium metavanadate have synergistic effect? What is Arsenic trioxide and sodium metavanadate's intrinsic molecular mechanism? Whether related to the change of protein O-glycosylation? To address these issues, this study focuses on the arsenic trioxide, sodium metavanadate, and two drugs induced HL-60 cells (human acute myeloid leukemia cell line) apoptosis, using a variety of molecular biology techniques to study the intrinsic molecular mechanism.Part I arsenic trioxide, sodium metavanadate induced HL-60 cell apoptosisMethods1.Use MTT assay to detect different concentrations of arsenic trioxide and / or sodium metavanadate on the HL-60 cell proliferation.2.Use flow cytometer to detect different concentrations of arsenic trioxide and / or sodium metavanadate on the HL-60 cells apoptosis.3.Use Hoechst 33258 and DNA ladder to detect different concentrations of arsenic trioxide and / or sodium metavanadate on the HL-60 cells apoptosis.Results1.MTT detected that different concentrations of arsenic trioxide can inhibit the proliferation of HL-60 cells. With the increase of drug concentration, the killing effect on cells also increased.2.Compared to the control group, the overall trend was that low concentration of sodium metavanadate inhibited the fatalities of cells and high concentrations promoted it.3.MTT detected that seperatly use of 5μmol / L arsenic trioxide and 10nmol / L sodium metavanadate on the HL-60, the inhibition of cell proliferation is less than the joint use of 5μmol / L arsenic trioxide and 10nmol / L sodium metavanadate.4.Flow results show that different concentrations of arsenic trioxide 20μmol / L, 10μmol / L, 5μmol / L, 1μmol / L added into HL-60 cells, after 24 hours later apoptotic percentage was 51.18%,24.88%,23.18%,19.19%. Different sodium metavanadate 500nmol/L,100nmol/L,10nmol/L,1nmol/L,1×10-2nmol/L added into HL-60 cells, after 24 hours later apoptotic percentage was 69.49%,55.18%,39.94%,31.87%,31.32%. 5μmol / L arsenic trioxide and 10nmol / L sodium metavanadate combined and added into HL-60 cells after 24 hours, apoptosis rate was 50.04%.5.Hoechest33258 staining. Fluorescence microscope detected that HL-60 cells were treated with different concentrations of arsenic trioxide and / or sodium metavanadate treatment, 24 hours later HL-60 cell nuclear integrity and chromatin evenly distributed, dyeing Talking in blank control group. In other experimental group nucleus dense stain, some experimental group chromatin condensation and fragmentation fracture, and even showed debris-like.6.HL-60 cells were treated with different concentrations of arsenic trioxide and / or sodium metavanadate, 24h later found 180-200bp or its integral multiple fragment, form a clear ladder.Part II arsenic trioxide, sodium metavanadate induced HL-60 cell apoptosis which associated with ppGalNAcT2Methods1.Use biological information to detect whether arsenic, vanadium can affect the activity of ppGalNAcT2.2.Using RT-PCR to detect different concentrations of arsenic trioxide, sodium metavanadate to the expression of ppGalNAcT2 at mRNA level. 3.Using immunofluorescence and flow cytometry to detect different concentrations of arsenic trioxide, sodium metavanadate to the expression of PNA.Results1.Successfully docking VO3 and ppGalNAcT2, As and ppGalNAcT2. VO3 can formate hydrogen bonds with different conditions of ppGalNAcT2. As only formed hydrogen bond with the activity related structures of ppGalNAcT2 at 2FFU. The results of molecular dynamics simulation confirmed that VO3 and As may affect the protein flexibility of ppGalNAcT2, especially the change of flexible ring B.2.RT-PCR results showed that added different concentrations of arsenic trioxide and / or sodium metavanadate into the experimental group, compared with the control group, at very low concentration of sodium metavanadate ppGalNAcT2's expression slightly increased at mRNA levels, at other experimental group ppGalNAcT2's expression were reduced at mRNA levels.3.Immunofluorescence results showed that the expression of peanut agglutinin PNA changed according to the concentration of arsenic trioxide and sodium metavanadate.4.Flow results showed that the expression of peanut agglutinin PNA increased with the increasing concentration of arsenic trioxide and sodium metavanadate. Only arsenic trioxide and sodium metavanadate in high concentration, as compared with the blank control group, the expression of peanut agglutinin PNA increased, and these changes have statistically significant.Part III Transfect ppGalNAc-T2 plus sense expression vector (pEGFP-C1-T2), ppGalNAc-T2 RNA Interferential Carrier (Si-T2) and Vacuity Carrier (pEGFP-C1, Scr-T2) into HL-60 cells, detected the changes of HL-60 cell's biological characteristicsMethods1.Transfected ppGalNAc-T2 (pEGFP-C1-T2) and pSilenCircle-T2 (Si-T2) into HL-60 cells, at the same time set up empty vector as control (pEGFP; pSilenCircle, Scr-T2), and blank control group (non-transfected HL-60 cells).2.Using RT-PCR to detect the expression of ppGalNAcT2 at mRNA level when different vector was transfected into HL-60 cells, and determining the transfection results.3.Using MTT to detect the proliferation capacity of HL-60 cell after transfectted with different vectors.4.through Internet to select tumor invasion and metastasis-related gene-MMP2, TGFβ1 which may be related to ppGalNAc-T2, using RT-PCR, Western blot to detect the expression of MMP2, TGFβ1 at mRNA level after transfected different vector into HL-60 cells.Results1.RT-PCR results showed that the experimental group which transfected with pEGFP-C1-T2, the expression of ppGalNAc-T2 at the mRNA level was significantly higher than the two control groups; the experimental group that transfected with Si-T2, the expression of ppGalNAc-T2 at the mRNA level was significantly lower than the two control groups.2.After transfected different vector into HL-60 cell, 48 hours later, cell's proliferative were inhibited at all experimental group, 144 hours later, the experiment group that transfected with pEGFP-C1-T2 HL-60 cells still showed growth inhibition , while the experiment group that transfected with Si-T2 HL-60 cell's proliferation was promoted.3.Through Internet to choose tumor invasion and metastasis-related gene-MMP2, TGFβ1 which may be related to ppGalNAc-T2. The expression of MMP2 and TGFβ1 at mRNA level were decreased after transfected with pEGFP-C1-T2. The expression of MMP2 and TGFβ1 at mRNA level were incresed after transfected with Si-T2.4.Using Western blot to detect the expression of MMP2 and TGFβ1, we found the expression of MMP2 and TGFβ1 decreased in the grouop of transfected with pEGFP-C1-T2, and the expression of MMP2 and TGFβ1 increased in the grouop of transfected with Si-T2.Part IV the effection of sodium metavanadate / arsenic trioxide on the HL-60 cell that Transfect ppGalNAc-T2 plus sense expression vector (pEGFP-C1-T2)Methods1.To detected the effection of sodium metavanadate / arsenic trioxide on HL-60 cell apoptosis of the expremient group that transfected with pEGFP-C1-T2 by MTT.2.Using RT-PCR, Western blot to detect the expression of tumor invasion and metastasis-related gene MMP2, TGFβ1 at mRNA and protein level.Results1.MTT showed that sodium metavanadate / arsenic trioxide could synergistic promote transfected pEGFP-C1-T2 HL-60 cell apoptosis.2.Compared with the blank control group, drug groups (sodium metavanadate / arsenic trioxide) and transfected with pEGFP-C1-T2, the effection of inhibit the expression of MMP2 and TGFβ1 at mRNA level was obvious. The result of Western blot was the same to the above results.ConclusionThis study is the first use arsenic trioxide and sodium metavanadate to treat HL-60 cell. We use different methods to detect whether sodium metavanadate and arsenic trioxide-induced HL-60 cell apoptosis correlation with Glycosyltransferase ppGalNAc-T2. Transfected ppGalNAc-T2 plus sense expression vector (pEGFP-C1-T2), ppGalNAc-T2 RNA Interferential Carrier (Si-T2) and Vacuity Carrier (pEGFP-C1, Scr-T2) into HL-60 cells, then use different methods to explore the relationship between the change of ppGalNAc-T2's expression and HL-60 cell's biological characteristics. We Added sodium metavanadate / arsenic trioxide into HL-60 cells which transfected with ppGalNAc-T2 plus sense expression vector (pEGFP-C1-T2). Then observe the action of three combine. According to the experimental results conclusions were drawn as follows:1.Arsenic trioxide inhibited the proliferation of HL-60 cell, and in time and measurement effects.2.Sodium metavanadate can inhibit the proliferation of HL-60 cells. Compared to the control group, the overall trend was that low concentration of sodium metavanadate inhibited the fatalities of HL-60 cells and high concentrations promoted the fatalities of HL-60 cells.3.Arsenic and vanadium can affect the active sites of Glycosyltransferase ppGalNAc-T2 thtough hydrogen bonds, affect ppGalNAc-T2's flexible structures, thus inhibiting the enzyme's catalytic function.4.Transfected ppGalNAc-T2 plus sense expression vector (pEGFP-C1-T2) into HL-60 cell can inhibit the proliferation of HL-60 cells. The expression of MMP2 and TGFβ1 was reduced in the cell that Transfected with ppGalNAc-T2 plus sense expression vector (pEGFP-C1-T2).5.Metavanadate sodium, arsenic trioxide and ppGalNAc-T2 plus sense expression vector (pEGFP-C1-T2), those have a synergistic effect.
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