| Objective: With the development of society,people gradually realize the negative effects of chemical synthetic drugs on human health and living environment;Returning to nature and protecting the environment has become a trend to resolve the relationship between human beings and environment.Due to the unique advantages of small side effects and from nature,natural medicine has attracted much attention from people.However,a large number of natural medicine has disadvantages such as unknown composition,single processing methods and the separation or purification technology is low.Most natural medicine and its mechanism is mostly unknown so that its application range is limited.In order to better use natural medicine,it is urgent to study the function mechanism of these drugs.In recent years it has been found that many secondary metabolites from plants and fungi have significant biological effect in anti-inflammatory effect,improving immunity and anti-tumor.It is named as iso-suillin an isomer of suillin isolated from Suillus flavus.After several years we found that iso-suillin showed strong biological activity effectively inhibiting the growth of tumor cell lines SMMC-7721,BGC,K562,A549 etc in vitro.In addition iso-suillin showed stronger inhibitory effect than cisplatin?CDDP?and 5-fluorouracil?5-FU?but no toxicity on normal human lymphocytes.Leukemia is a kind of malignant clonal disease for hematopoietic stem cells.Because of uncontrolled proliferation and impaired differentiation,leukemia cells accumulated in bone marrow and other hematopoietic tissues to inhibit normal hematopoietic function and infiltrate other non hematopoietic tissues.The incidence of leukemia was sixth in a variety of tumors in China.Although there was a certain effect on leukemia with the treatment of varioustherapy,but the leukemia had still a very high mortality rate.So it is an urgent task to seek medical treatment for leukemia.After research of many years we had found that there was a obvious inhibition on K562 cells induced by iso-suillin through mitochondrial apoptosis pathway and death receptor pathway,but the inhibition on the proliferation of K562 cells and its molecular mechanism were uncovered in public.On the basis of previous research,this research would make efforts to in-depth exploration of the molecular mechanism in which iso-suillin induced the proliferation inhibition in K562 cells.By revealing the molecular mechanism of iso-suillin induced K562 cell proliferation inhibition,we hope find the key target specific killing leukemic cell or even provide theoretical basis and possible drug treatment for leukemia.Methods:1 Cell cultureK562 cells were cultured with DMEM medium supplemented with 10%fetal bovine serum,1% myllicin mixtureat in an incubator containing at 37 ?with 5% CO2.2 The effect of iso-suillin on gene expression in K562 cells and bioinformatics analysisTotal m RNA was extracted after the treatment of iso-suillin.when RNA quality detection was acceptable,we used gene chip to detect the whole genome expression profiles of treatment group and control group.Then we had an enrichment analysis on differential expression gene between control group and iso-suillin treatment group treated with iso-suillin include KEGG pathway enrichment analysis and Go term enrichment analysis of molecular function?MF?,biological processes?BP?and cellular components?CC?.3 The effect of iso-suillin on inhibition in K562 cells detected by MTTThe K562 cells in exponential growth phase were planted into 96-well multiplates and treated with iso-suillin in different concentrations for 24 hour,incubated with MTT for 2-4 hours then added DMSO to dissolve violet crystal.The proliferation activity was measured by the Microplate Reader and drawnthe cell proliferation inhibition curve.4 The effect of iso-suillin on the proliferation of K562 detected by soft agar clone formation assayThe K562 cells in exponential growth phase were planted into 6-well plates with iso-suillin in different concentrations for 24 hour.Then 800 cells one bottle had been planted into culture flask?2.5cm2?until clones became highly visible.Each flask was filled with 1 ml soft agar?1.2%?.Freezed in ice,stained with crystal violet for 5 min,washed with PBS buffer two times and measured the clone number and size.5 The effect of iso-suillin on the cell cycle distribution in K562 cells by flow cytometryThe K562 cells in exponential growth phase had been planted into 6-well plates in a concentration of 1 × 105/ml,with iso-suillin in different concentrations for 24 hour.Collectted cells,washed two times with precooling PBS buffer,then we fixed the cells with 70% ethanol over night?firstly using300 ?l PBS to resuspense the cell,then slowly dropping 700 ?l ethanol?.The cells were collected and washed two times with PBS.At last added 500 ul PI staining solution?PI 50 ug/ml with RNAase?to each cell sample and incubated at 37 ? for 30 min in the dark.We used flowjo7.6.5 to analyze the distribution of cell period.6 The effect on relative protein esspreesed in K562 treated with iso-suillin detected by Western BlotControl and iso-suillin-treated cells were washed twice with precooling PBS,and then lysed in appropriate extraction buffer comprised of RIPA,protease inhibitor mixture and phosphatase inhibitor on ice for 30 min.Protein concentration was determined by ND2000 C.Then centrifuged at 14000 rmp for 20 min.After added loading buffer,the supernatant had a incubation of boiling water for 5 min and stored at-20 C for a long time.Proteins were loaded at concentration of 50 ug per lane and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis?SDS-PAGE?.Thereafter,the protein was electrotransferred onto 0.22 ?M polyvinylidene difluoride?PVDF?membrane.Membrane was blocked in blocking buffer containing 5%nonfatmilk for 2.5 h.Primary Antibodies for ?-actin,?-H2 AX,cyclin D1,CDK4,E2F-1,p-Rb,PCNA,p21,p38 and p-p38 were incubated overnight at4°C.Membrane was washed by Tris-buffered saline containing 0.1% tween-20 surfactant and soaked in correspondent fluorescently-labeled secondary antibodies at room temperature for 1 h and analyzed using Odyssey Clx.?-Actin was used as the internal control.7 The verification of p53 gene mutation in K562 cellsFollowing the extraction of total DNA in K562 cells in convention al method,amplification with the F-p53 5?-CATCGCTATCTGAGCAG CGCTCA-3?and the R-p53 5?-TCTGGGCTTCTTGCATTCTGGGAC-3? primers,1% agarose gel electrophoresis at 100 V for 20 min and g el extraction and sequencing we checked the p53 m RNA sequences in K562 cells by DNAMAN with the wild type p53 gene sequence from NCBI website.8 Effect of P38 inhibitor on cell proliferation related factor induced by iso-suillin in K562 cellsFirst of all,K562 cells should be treated with 0.5 ?M?IC50?SB203580for 30 min before treatment of iso-suillin.MTT assay,Soft agar colony forming assay,flow cytometry and western blot were simillar to above steps.9 Statistical analysesThe results were expressed as mean ± standard deviation evaluated with SPSS 13.0 by analysis of One-Way ANOVA.When P<0.05,it was considered statistically significant.Results:1 The effect of iso-suillin on K562 gene expression and bioinformatics analysisCompared with the control group?C?,the treatment group?T?had 639 differential expression genes,including 443 up-regulated and 196 down regulated.The up-regulated genes mainly were ATF3,GFPT1,S100 P,PHGDH,ATP2A3,NRCAM,SESN2,ATF5,LAMP3 and LAD1.Furthermorethere were a number of genes associated with apoptosis,such as tumor necrosis factor gene?TNFAIP3,TNFRSF9,TNFRSF10 B,TNFRSF 11B?,DNA damage inducible transcription protein gene DDIT4 and differentiation factor GDF15.The down-regulated genes included MFAP3,LTNFRSF10 B,SH3BP2,RUNDC3 B,DIP2C,SLC7A11,WARS,AAK1,ENAH,LPGAT1 and EPAS1.And mitogenic signal composition RAS related genes?REM1,RAB15?were down-regulated.It maybe related to the inhibitionon K562 cells induced by iso-suillin.Further screening we found that the cell cycle kinase inhibitor CDKN1A?p21?was significantly up-regulated correspond to the result of Western blot assay.GO term analysis showed that the differential genes were mainly distributed in the cytoplasm,cytoskeleton,transport vesicles,lysosomes;its molecular function mainly include vitamin?coenzyme?,enzyme binding and neutral amino acid transmembrane transporters.The analysis of the KEGG pathway showed that there were a number of signaling pathways associated with differentially expressed genes and we identified the most likely?P-value was minimum?pathways according to the differential expressed genes as follow.P53 signaling pathway,insulin signaling pathway,a carbon pool of folic acid,alanine,aspartic acid and glutamic acid metabolism,nitrogen metabolism and aminoacyl t RNA biosynthesis and those P-values were 0.0004,0.0055,0.0105,0.0131,0.016,0.0352 and 0.0403.-lin?P-value?were 1.31?0.49?0.41?0.43?0.44?0.25?0.25.These differentially expressed genes mainly enriched in insulin signaling pathway and p53 signaling pathway.The insulin signaling pathway involved in a classical MAPK signaling pathway.The MAPK signaling pathway may play an important role in genes differential expression induced by iso-suillin as p53 gene was mutation in K562 cells.2 The effect of iso-suillin on inhibition in K562 cells detected by MTTK562 cell proliferation inhibition rate was increased with the increasing of the concentration of iso-suillin.Proliferation inhibition rate was 0.28 ± 4.21,0.16 ± 2.59,11.23 ± 8.33,29.32 ± 2.35?%?and the correspondingconcentration of iso-suillin was 0,1.37,2.74,5.48,10.95 ?M.The results showed that iso-suillin could inhibit K562 cells proliferation in vitro in a concentration dependent manner.3 The effect of iso-suillin on the proliferation of K562 detected by in soft agar clone formation assayHad treated with the iso-suillin in concentration of 0,2.74,5.48,10.95?M for 24 h,the K562 cells clone formation rates were 39.08 ± 13.13,25.8 ±11.91,15.75 ± 5.25,11.67 ± 8.80?%?;clone diameters were 1.88 ± 0.17,1.88± 0.30,1.15 ± 0.07,0.77 ± 0.30?mm?.The experimental results show that with the increasing of iso-suillin concentration,colony formation was significantly reduced and diameter became shorter.It suggested that iso-suillin has obvious inhibitory effect on the proliferation of K562 cells.4 The effect of iso-suillin on cell cycle of K562 cellsK562 cells had been treated with iso-suillin in concentration of 0,2.74,5.48,10.95?M for 24 h,then cells proportion of the total cell in G0/G1 phase accounted for 48.31 ± 2.52,60.27 ± 3.67,61.07 ± 3.59 and 67.70 ± 2.19?%?;S phase cells were 34.48 ± 1.57,17.98 ± 3.09,21.33 ± 5.89 and 18.18 ± 7.58?%?;G2 phase cells were 17.22 ± 1.76,21.74 ± 0.59,17.60 ± 2.60 and 14.12 ±5.56?%?.With the concentration increasing the proportion of G0/G1 phase cells had been increasing significantly?P<0.02?;S and G2 proportion had been decreaseing.It was statistical significance?P<0.02?.It suggests iso-suillin could induce G0/G1 block phenomenon in K562 cells.5 Detection of proliferation related protein expression by Western blotK562 cells had been treated with 0,2.74,5.48,10.95 ?M iso-suillin for24 hour.Western blot detected related protein expression and phosphorylation.The results were as follows: compared with ?-actin protein as reference,the relative content of cyclin D was 0.8072 ± 0.0134,0.8098 ± 0.0431,0.7388±0.0594,0.6954 ± 0.0296;CDK4 was 0.0216 ± 0.0003,0.0202 ± 0.0009,0.0095 ± 0.0006,0.0038 ± 0.0004;PCNA was 0.1256 ± 0.0027,0.0573 ±0.0016,0.0505 ± 0.0026,0.0468 ± 0.0013;p-RB was 1.4254±0.0358,0.7835±0.0485,0.8632±0.0399,0.2117±0.0276;E2F-1 0.4987 ± 0.0423,0.2754 ± 0.0099,0.2568 ± 0.0119,0.0419 ± 0.0005;p21 0.0646 ± 0.0032,0.1337 ± 0.0013,0.1286 ± 0.0009,0.1228 ± 0.0019;p38 0.7641 ± 0.0149,0.8479 ± 0.0251,0.7765 ± 0.0198,0.8378 ± 0.0667;p38?* hosphor Thr180/Tyr182?0.1859 ± 0.0025,0.1144 ± 0.0052,0.0982 ± 0.0061,0.0911 ±0.0113.With the increasing of concentration in K562 cells treated with iso-suillin,CDK4,cyclin D protein,PCNA and E2F-1 were down-regulated?P<0.05?;the content of cell cycle proteins depend kinase inhibitor protein p21 was up-regulated significantly.It is strange that the phosphorylation of p38 decreased?P<0.05?,later found that the activation of p38 is in the early,but quickly degradation.K562 cells were treated with 5.48 ?M for 0 h,3 h,6 h,12 h,24 h.Western blot measured the relative content of p-p38 is 0.0050 ± 0.0003,0.0411 ± 0.0020,0.0133 ± 0.0005,0.0036 ± 0.0013,0.0103 ± 0.0006;the relative content of ?-H2 AX was 0.0295 ± 0.0032,0.0646 ± 0.0032,0.1337 ±0.0013,0.1286 ± 0.0009,0.1228 ± 0.0019.The results showed that treated with iso-suillin,the phosphorylation level of p38 increased at first and then dropped in K562 cells.The highest relative content was at 3 hour.The content of ? –H2AX increased gradually and it was different significantly after 6 hour.6 The verification of p53 gene mutation in K562 cellsThe CDS sequence of p53 protein in K562 cells has a shift mutation?406407,ins C?,leading to the termination codon appear in advance;Western blot was unable to detect the normal expression of p53 protein nor any p53 mutants.7 The effect of iso-suillin on the proliferation of K562 cells after treated with p38 inhibitorProliferation inhibition rates of K562 cells severally were 0,6.02 ± 3.63,20.35 ± 3.78,36.33 ± 15.76?%?in the control group?blank controller?and SB203580,iso-suillin + SB203580,iso-suillin treated groups;clone formation rates were 36.83 ± 4.09,17.33 ± 11.7,7.58 ± 2.48,2.71 ± 1.87?%?,clone diameter were 2.42 ± 0.50,3.07 ± 0.49,3.89 ± 0.84,2.44 ± 0.51?mm?.Treatment groups showed higher degrees of inhibition compared with controlgroup,but the proliferation inhibition rate in SB203580+iso-suillin group was significantly reduced compared with the iso-suillin group.It was statistical significance?P<0.05?.K562 cells cycle distribution were measured by flow cytometry in the control group?blank controller?and SB203580,iso-suillin+SB203580,iso-suillin groups.The proportions of G0/G1 phase were39.26 ± 1.17,43.53 ± 0.97,44.00 ± 1.24,65.22 ± 4.74?%?;S were 32.26 ±2.60,25.14 ± 2.82,26.43 ± 4.21,18.95 ± 3.47?%?;Cell ratios of G2 were28.47 ± 1.43,31.33 ± 1.86,29.58 ± 2.98,15.83 ± 1.28?%?.It suggested that SB203580 could reduce obviously G0/G1 arrest induced by iso-suillin in K562 cells.SB203580 is an inhibitor of p38,so it was concluded that p38 signaling pathway may play an important role in inhibiting the proliferation induced by iso-suillin in K562 cells.8 The effect of iso-suillin on the related protein expression in K562 cells treated with p38 inhibitorThe relative content of p21 were 0.4984 ± 0.0087,0.3296 ± 0.0603,0.2091 ± 0.0183,1.1401 ± 0.0233 in the control group?blank controller?and SB203580,iso-suillin SB203580,iso-suillin treatment groups;The relative content of p-p38 protein were 0.3300 ± 0.0052,0.1298 ± 0.0163,0.1298 ±0.0164,0.1205 ± 0.0054,0.6562 ± 0.0312.It was found that after the addition of p38 inhibitor SB203580,the level of p38 phosphorylation and the relative content of p21 decreased significantly compared with control group,and the p38 phosphorylation and p21 content in SB203580+iso-suillin treated group was significantly lower than in the control group.It was statistical significance?P<0.05?.Conclusions:1 Iso-suillin can inhibit the proliferation of K562 cells in vitro.2 Iso-suillin can induce G0/G1 cell cycle arrest in K562 cells.3 The different expression genes after treatment with iso-suillin in K562 cells had a significant enrichment in p53 signal pathway and p38 MAPK signal pathway.4 P38 MAPK is a biological switch mediating cell cycle arrest induced by iso-suillin in K562 cells. |
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