| ObjectiveColorectal cancer(CRC)is the third most frequently diagnosed cancer worldwide.A total of 132,700 new CRC cases and 49,700 CRC deaths are projected to occur in the United States in 2015.Surgery remains the curative option of choice.However,still 40%-50%of CRC patients died of the disease within five years of diagnosis.Mortality of colorectal cancer in 2005 increased 70.7%more than that in 1991,with an average annual increase of 4.71%.In 2005 the incidence and deaths number of colorectal cancer wrere spectively 172,000 and 99,000,more than the United States.With the improvement of living standards and changing diets,the incidence of colon cancer in Chinese cities showed an increasing trend.Early surgical treatment of colorectal cancer is preferred,but there are no significant symptoms in early stage of colorectal cancer,so 40%of colorectal cancer patients to be diagnosed are already advanced.Therefore,chemotherapy and drug treatment for colon cancer patients can prolong life cycle after surgery and improve quality of life.Currently commonly used drugs in colorectal cancer,which have different targets and mechanisms of action,generally induce tolerance of tumor cells.Therefore,the discovery of new low toxicity,efficient and inexpensive drugs is very necessary.Over time,tumor cells create emergency mechanism to escape from death,eventually produce drug tolerance.In the case of the current surge in drug-resistant tumors,developing an anti-tolerance drug of CRC and explaining its mechanism of action has an important scientific value and clinical significance.Arenobufagin is extracted from the inside of one toad poison toad venom compound cool.Studies have shown that Arenobufagin may be targeted PI3K/Akt/mTOR regulation of autophagy and apoptosis of hepatoma cell interaction,inhibit adhesion,migration and invasion of HepG2 cells.Arenobufagin can inhibit human umbilical vein endothelial cell proliferation,and inhibiti angiogenesis in chick chorioallantoic membrane.But the cell death mechanism of Arenobufagin induction in colon cancer is not clear.In view of this,the subject of Arenobufagin induction apoptosis in colorectal cancer and its regulatory mechanisms were studied.Drp1 can induce the dephosphorylation by PGAM5,thereby regulating mitochondrial fission.Studies have shown that inhibition of cytokine signal 6 can cause PGAM5 and Drpl complex formation,reduction of Drpl phosphorylation increase its induced mitochondrial rupture,which can promote apoptosis,increase Bax conformational change and target to mitochondria and oligomerization.This study was designed to study Arenobufagin effect on colon cancer,whether was by internal Bax-PGAMSL-Drpl complex formation induction apoptosis in colon cancer.Changes of apoptosis was compared in HCT-116 WT(wild type)cells and HCT-116 Bax-/-cells at PGAM5 of 3,-PGAM5L and PGAM5S different gene silencing conditions.PGAM5L-Bax-Drpl interaction were confirmed by Co-precipitation through immunization,between.In vitro and in vivo experiments,we were applied staurosporine and cisplatin,further confirmed the important role of Bax-PGAM5L-Drp1 complex in endogenous apoptosis.Interpretation of the molecular mechanisms of Arenobufagin to induce apoptosis in colon cancer,can provide site directed for clinical treatment.Methods1.Effect of Arenobufagin on colon cancer cell viability SW480,DLD1,LS174T three kinds of colon cancer cells,37 ℃ 5%CO2 culture.The cell density was adjusted to approximately 10,000/well(96 well plates).The adherent cells were added to a concentration gradient of Arenobufagin,each hole 100μl.Each concentration included 5 wells,while setting up the control group.Respectively,after 6h,12h,24h,48h drug intervention,each well were added 5mg/mL MTT 20μL,and cultured 4h.Discard the old solution,and add to each well 150μL dimethyl sulfoxide.Then set shaker 10min,to purple crystals completely dissolved.Enzyme-linked immunosorbent assay measured OD by 490nm.Cell survival= test group OD/control OD values.The experiment was repeated three times.2.Hoechst 33342/PI observed colon cancer cell morphology Cell culture methods see Method 1 plank.The adherent cells were added to a concentration gradient of Arenobufagin,each hole 100μL.Each concentration included 3 wells,while setting up the control group.They were cultured 6h,12h,24h after administration.20mg/ml Hoechst 33342 were prepared to 5-10μg/ml fluid storage solution by PBS.Adherent cells medium was discarded,and cells were washed with PBS 1-2 times.Each well was added 35μl Hoechst 33342 working solution and incubated at 37 ℃ 15-20 min.Hoechst 33342 was abandoned and washed 1-2 times by PBS.The experiment was repeated three times.3.Annexin V-FITC/PI double staining detected apoptosis rate by flow cytometry Cell culture methods see Method 1 plank.Cell culture see Methods 1.The adherent cells were added to a concentration gradient of Arenobufagin each hole 1mL.After the establishment of the control group,each concentration was intervened,according to Annexin V/PI flow kit instructions,using the U.S.company EPICS XL Beckman analytical sample measured by flow cytometry,according to Annexin V-FITC(X)/PI(Y-axis)to make the number of fluorescence scattergram obtained by the two parameters in 4 quadrants:the upper left quadrant(K1)cells to damage generated during collection,the upper right quadrant(K2)is late apoptotic and necrotic cells,the lower left quadrant(K3)for the live cells,the lower right quadrant(K4)early apoptotic cells,number of cells in each quadrant is the total number of cells in the subject proportion.The experiment was repeated three times.4.Western blot analysis of the effects of parthenolide on caspase-3 protein expression.Colon cancer cells in kinds of 1 × 105/well were seeded in 6-well plates,the adherent cells were added to a concentration gradient of Arenobufagin,each hole 2ml.After the establishment of the control group,each concentration was intervened.Use Bradford protein quantification method,take 80p.g protein samples and add five-times sample buffer on 12%SDS-PAGE.By he wet transfer method,protein transfereed to a PVDF membrane,5%skim milk at room temperature closed 1h.Incubating overnight at 4℃ by primary antibody,next day the membrane was incubated by HRP-conjugated appropriate secondary antibody(1:1000 dilution)at room temperature for 1h.Then we used KODAK Image Station 4000MM Digital Imaging System chermiluminescence assay.Image exposure of protein bands obtained was analyzed by the gradation Molecular Imaging Software Version 4.0.5.Immunofluorescence.Cells were incubated with antibody at 4 ℃ overnight,incubated with Cy3-labeled goat anti-rabbit or anti-mouse IgG antibody(1:500)in darkness for 60 min at room temperature,and then counterstained with 4’6-diamidino-2-phenylindole(DAPI),and examined under confocal microscope(Nikon,Japan)with excitation and emission wavelengths of 550 and 570 nm,respectively with a 100×1.40NA oil immersion objective.For mitochondrial staining,200 nM MitoTracker Green(Molecular Probes)was added to cultures 30 min before fixation.6.Lentivirus interference different protein gene expression in the growth of colon cancer cells The differential protein gene shRNA lentiviral infection to colon cancer cells were stably transfected with shRNA,which influenced on the growth of colon cancer cells.7.JC-1 mitochondrial membrane potential assay Cells were resuspended,stained by 0.5 ml JC-1 working solution,and added 250 μl 1X buffer.FACS Calibur flow cytometry analysis software with CellQuest relative intensity of fluorescence observed the changes of mitochondrial membrane potential8.Mitochondrial morphology analysis(Micro-P).Cells were stained with MitoTracker and cell images were taken with confocal microscope.Cells were then analyzed by Micro-P software.Micro-P software measured individual mitochondrion according to the area,axial,and length/width,and sorted mitochondria into five types.9.TEM cell ultrastructure The cells were collected,1000 rpm centrifuged for 5 minutes,4 ℃ PBS and resuspended 2000 rpm for 5 minutes.Precipitation with 2.5%glutaraldehyde solution at 4 ℃ overnight,drained fixative with 0.1 M,pH 7.0 phosphate buffer of rinse the sample three times,and embed good sample.Samples microtome in 70～90nm chips,the sections were lead citrate,uranyl acetate solution,and 50%ethanol solution of saturated stained 15 min,TEM observation.10.Immunoprecipitation.Cell extract was mixed with anti-PGAM5 and anti-Drp1 agarose beads in a ratio of 1 mg of extract per 30 ml of agarose at 4℃ overnight.The beads were then pelleted at 2,500 g for 3 min and washed with lysis buffer five times.The beads were subjected to elution with 5 vol of 0.5 mg/ml peptide for 4 hr or directly boiled in 1 × SDS loading buffer.11.Orthotopic CRC model.SW480-eGFP(5×106)cells were injected subcutaneously to BALB/c-nu.Ten days later.Tumor tissue was divided into small pieces,approximately 1 mm3.Nude mice were anesthetized,and colon surgical orthotopic implantation(SOI)of tumor was performed.Animals were kept in a sterile environment.To investigate tumor metastasis,various organs were collected from the tumor-bearing mice.12.Xenograft CRC model HCT-116 and HCT-116 Bax-/-cells(1 × 107)were inoculated subcutaneously on the groin of nude mice and allowed to grow for-7 days to reach a tumor volume of-50 mm3.Their body weights and tumor volumes were measured every 5 days throughout the treatment period.The mice were killed at the end of the experiments.Tumor xenografts were removed and weighed.13.Statistical analyses.Using SPSS 13.0 statistical software for analysis,results are expressed as:mean + standard deviation.Between the two groups were compared using independent samples t test.Among groups were compared using One Way-ANOVA,homogeneity of variance using LSD method,using the Welch method variance arrhythmia corrected pairwise comparisons using Dunnett T3 method;P<0.05 indicates a statistically significant.Results1.Arenobufagin induces tumor cell apoptosis.To address the role of arenobufagin on cell viability,various CRC cell lines,including SW480,DLD-1 and LS174T,were tested(P<0.01).Arenobufagin decreased cell viability both in a dose-and time-dependent manner.We then examined which cell death subroutine was responsible for the lowered viability.Rounding-up of the cells,retraction of pseudopodes,reduction of cellular and nuclear volume(pyknosis)and nuclear fragmentation(karyorrhexis)in arenobufagin treated SW480 cells suggested the morphological features of apoptosis.Hoechst 33342 staining,annexin V/7-amino-actinomycin D double staining showed that most of the cell death induced by arenobufagin can be classified as apoptosis both in SW480 and DLD-1.Activation of caspases is a biochemical feature of apoptosis.Immunoblotting assessment showed that caspase 9 was cleaved by arenobufagin.Activated caspase-9 in turn cleaves and activates caspase-3.The cleaved caspase 9 and caspase 3 were increased by arenobufagin in a dose-dependent manner.The cleavage of PARP,a caspase-3/7 substrate,was also increased by arenobufagin treatment.The apoptosis caused by arenobufagin were efficiently abrogated by pretreatment with Z-VAD-fmk(t= 16.00,P<0.001),a broad spectrum caspase inhibitor,suggesting that arenobufagin induced cell death was caspase-dependent(t=-14.59,P<0.001).These morphological and biochemical changes suggest that the cell death caused by arenobufagin is apoptosis.2.The intrinsic apoptosis caused by arenobufagin is Bax-dependent.Arenobufagin induced the translocation and accumulation of Bax in the mitochondria in SW480 and HCT116 cells.Dimers were formed in a dose-dependeng manner with the treatment of arenobufagin.We then tested if the arenobufagin induced apoptosis is Bax-dependent with HCT116(WT)and HCT116 Bax-/-cells.Arenobufagin significantly increased the apoptosis rate(P<0.01)and decreased the cell viability(P<0.01)in HCT116 WT cells than that of HCT116 Bax-/-cells in a dose dependent manner.Cleavage of PARP and caspase 9 were observed in HCT116 WT cells accompanied by Bax activation.The concentration-dependent shift in the emission spectrum from red to green measured by flow cytometry indicated the dissipation of△Ψm by arenobufagin.As expected,arenobufagin promotes the release of cyto C from mitochondria to cytosol in HCT116 WT cells as compared to HCT116 Bax-/-cells.Translocation of AIF from mitochondria to nuclear was also observed in HCT116 WT cells.Immunofluorescent staining provided direct evidence that arenobufagin drives AIF shifting to the nuclear with Bax punctae in the mitochondria.This suggests that arenobufagin has a strong permeabilizing effect on mitochondrial membranes.Thus,the robust arenobufagin-induced release of cyto C and AIF from mitochondria indicates that it not only has a strong permeabilizing effect on outer mitochondria membrane,but also has efficient effect in disrupting the inner mitochondrial membrane integrity.3.PGAM5L is required for Bax-mediated intrinsic apoptosis.We then tested their roles in arenobufagin induced intrinsic apoptosis.Small interfering RNA(shRNA)oligos targeting the 3’-untranslated region of PGAM5L,but not of PGAM5S increased the viability(SW480:1μM:P<0.001;5μM:P=0.001;HCT116(WT):1μm:P<0.001,5μM:P<0.001)and decreased the apoptosis rate(SW480:P<0.001,HCT116:P=0.001)in SW480 and HCT116 WT cells treated with arenobufagin.As expected,both PGAM5L shRNA and PGAM5S shRNA had no effects in HCT116 Bax-/-cells treated with arenobufagin.We next asked if PGAM5L is needed in the translocation of Bax.The overlap between mito-tracker(green)and Bax staining(red)which showed yellow fluorescence(merge)indicates that arenobufagin induced relocation of Bax to mitochondria in SW480 cells.PGAM5L shRNA,but not PGAM5S shRNA,inhibited the translocation of Bax induced by arenobufagin.The cleavage of PARP was also inhibited by transfection of PGAM5L shRNA,but not of PGAM5S shRNA.Taken together,our findings demonstrate that PGAM5L is required for arenobufagin induced intrinsic apoptosis mediated through Bax translocation to mitochondria.4.Drpl is also needed in the induction of intrinsic apoptosis process.Drpl siRNA or Drpl inhibitor MDIVI-1 significantly recovered the viability of both SW480 and HCT116 WT cells that were lowered by 1μM and 5μM arenobufagin treatments(SW480:F=22.16,P=0.002;HCT116:F=24.07,P=0.017).The arenobufagin induced apoptosis was significantly reduced by Drpl siRNA or MDIVI-1(SW480:F=19.13,P=0.020;HCT116:F=10.27,P=0.012).The lowered expression of Drpl by arenobufagin together with its activation of Bax were interfered by PGAM5L shRNA,but not by PGAM5S shRNA.Cell viability(IμM:F=2.68,P=0.147;5μM:F=1.63,P=0.273)and apoptosis rate were not affected in HCT116 Bax-/-cells with or without arenobufagin treatment,nor are they affected by PGAM5L shRNA or PGAM5S shRNA.Immunofluorescence staining showed that the accumulation of Bax in mitochondria induced by arenobufagin was severely disrupted by Drpl siRNA or MDIVI-1 in SW480 cells.Drpl siRNA or MDIVI-1 also significantly lowered the cleavage of PARP.We quantified PGAM5 and Drpl mediated mitochondrial morphological changes using a recently developed softwar,Micro-P.The number of small fragmented mitochondria(type 1)was significantly increased and the number of normal mitochondria was decreased in cell treated with arenobufagin.PGAM5L siRNA(P=0.005),but not PGAM5S siRNA(P=0.180)alleviates the increasing of the type 1 mitochondria induced by arenobufagin.Drpl siRNA and MDIVI-1 treatment displayed a significantly higher percentage of type 5 mitochondrion(F=10.44,P=0.003;Drp1 siRNA:P=0.003;MDIVI-1:P=0.003),with a concomitant decrease in type 2(F=7.58,P=0.009;Drpl siRNA:P=0.01;MDIVI-1:P=0.008).These results suggested that interaction between PGAM5L and Drpl is linked to mitochondria fission.5.Bax Interacts with PGAM5-Drpl Complex.To find out if the specific association between Drpl and PGAM5 is required for the induction of intrinsic apoptosis,co-immunoprecipitation was performed in HCT116 WT and HCT116 Bax-/-cells.When immunoprecipitated with PGAM5 antibody,a strong interaction between Bax,Drpl and PGAM5 was found in HCT116 WT under apoptotic condition.The interaction was nearly completely abolished in HCT116 Bax-/-cells under both normal or apoptotic conditions.To further confirm the result,we then tried to rescue apoptosis by transfection with a HA-tagged Bax coding sequence into HCT116 Bax-/-cells.As expected,the interaction between Bax,Drp1 and PGAM5 was restored.Also,iMAC2 attenuated the interaction between Drpl and PGAM5 in apoptotic HCT116 WT cells.These results suggest that a Drpl-PGAM5 complex was formed in a Bax dependent manner.PGAM5L shRNA,but not PGAM5S shRNA,almost completely abolished the interaction between Drp1 and Bax.When PGAM5L expression was restored,the interaction was re-established.The interaction between PGAM5,Drpl and Bax was confirmed in SW480 cell line.The immunoprecipitated Bax and PGAM5 increased in a time dependent manner as baited with Drpl under arenobufagin treatment.A time dependent dephosphorylation of Drpl Ser637 was observed in arenobufagin treated HCT116 WT cells,but not in HCT116 Bax-/-cells.The dephosphorylation of Drpl Ser637 was positively correlated with the expression of Bax.These results further strengthen our results that Bax activation and Drp1 dephosphorylation are indispensible for the formation of the Bax-PGAM5L-Drpl complex in the process of intrinsic apoptosis.6.Animals.Arenobufagin decreases the growth and metastasis of CRC by inducing Bax mediated apoptosis.To further investigate if aenobufagin induced intrinsic apoptosis is Bax-dependent in vivo,heterotropic CRC tumors derived from HCT116 Bax+/+ and Bax-/-cells were xenografted to BALB/c-nu mice.The death rate and body weight loss caused by arenobufagin are lower than that of caused by cisplatin in both HCT116 Bax+/+ and Bax-/-tumor bearing mice.Both arenobufagin and cisplatin decreased the tumor volume and tumor weight as compared to the control group.Arenobufagin decreased tumor weight and tumor volume(weight:t=-4.10,P=0.006;volume:t=-11.07,P<0.001)of Bax+/+ tumor as compared to the Bax-/-group.Cisplatin also decreased tumor weight and tumor volume(weight:t=-3.06,P=0.022;volume;t=-3.53,P=0.033)of Bax+/+ tumor as compared to the Bax-/-group.When baited with PGAM5,Drpl and Bax were immunoprecipitated from arenobufagin and cisplatin treated Bax+/+ tumor tissues extracts.The in vivo formed complex might be Bax-PGAM5L-Drp1,but not Bax-PGAM5S-Drpl.Conclusions Arenobufagin decreases the growth and metastasis of CRC by inducing Bax-PGAM5L-Drpl complex mediated intrinsic apoptosis. |
PDF Full Text Request