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Objective To compare the differences in the anti-tumor growth effects of organisms with different injections of CT26 tumor cell RNA loaded into nanoliposomes. Methods The extracted tumor RNA was loaded into nanoliposomes to prepare tumor RNA nanoliposome vaccines, and the related properties of nanoliposome vaccines were investigated. The particle size of nanoliposome vaccines was （120.0±12.1）nm and zeta potential was （3.39±0.56）mV. Tumor RNA nanoliposome vaccines were injected into different parts of the mice to test and analyze the influence of different injections on the growth of colon cancer transplanted tumors in mice. Results Tumor RNA nanoliposome vaccines were used to inject tumor-transplanted mice in different ways. Compared with underarm injection, intraperitoneal injection enhanced the organism's anti-tumor immune response and inhibited the growth of transplanted tumors more effectively. The H&E staining of important organs in mice was compared and no obvious organic lesions were found in the organs. Conclusion Intraperitoneal injections of nanoliposome loaded with tumor RNA can enhance the body's anti-tumor immune response more effectively than underarm injections.
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Objective To explore a newly discovered transmembrane protease serine 4 (TMPRSS4) isoforms and its molecular charcteristics in transfected colon cancer cells. Methods The named T4-1A and T4-1B of the TMPRSS4-isoforms were authenticated by the RT-PCR and Western blot,and then transfected to human colon cancer cells (DLD-1). Those stable transfected cells of migration and invasion were illustrated using wound healing assays and matrigel invasion assays. Results Successfully constructed T4-1A and T4-1B recombinant vectors,and obtained T4-1A and T4-1B transfected cell lines,and their T4-1A and T4-1B were highly expressed in stable DLD-1. Compared to cells transfected with empty vector of pcDNA6,the transfected DLD-1 with T4-1A enhanced migration and invasion were statistical significance (P < 0. 05). However,compared to pcDNA6 no significant1 difference was found for T4-1B. Moreover,the biological characteristics of T4-1A and TMPRSS4 were very similar. Conclusion The T4-1A and T4-1B is newly TMPRSS4-isoforms,and protease domain included to a T4-1A has further facilitated the migration and invasion of colon cancer cells,and further studies provide a theoretic base in the molecular biomedical characteristics of TMPRSS4.
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OBJECTIVE@#To explore the role of miR-593 in regulating the proliferation of colon cancer cells and the molecular mechanism.@*METHODS@#Bioinformatics analysis identified PLK1 as the possible target gene of miR-593. Luciferase assay was employed to verify the binding between miR-593 and PLK1, and qRT-PCR and Western blotting were used to verify that PLK1 was the direct target gene of miR-593. CCK-8 assay was performed to test the hypothesis that miR-593 inhibited the proliferation of colon cancer cells by targeting PLK1.@*RESULTS@#Luciferase assay identified the specific site of miR-593 binding with PLK1. Western blotting showed a significantly decreased expression of PLK1 in the colon cancer cells transfected with miR-593 mimics and an increased PLK1 expression in the cells transfected with the miR-593 inhibitor as compared with the control cells ( < 0.05). The results of qRT-PCR showed no significant differences in the expression levels of PLK1 among the cells with different treatments ( > 0.05). The cell proliferation assay showed opposite effects of miR-593 and PLK1 on the proliferation of colon cancer cells, and the effect of co-transfection with miR-593 mimic and a PLK1-overexpressing plasmid on the cell proliferation was between those in PLK1 over-expressing group and miR-593 mimic group.@*CONCLUSIONS@#miR-593 inhibits the proliferation of colon cancer cells by down-regulating PLK1 and plays the role as a tumor suppressor in colon cancer.
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Humans , Binding Sites , Cell Cycle Proteins , Genetics , Metabolism , Cell Line, Tumor , Cell Proliferation , Colonic Neoplasms , Metabolism , Pathology , Down-Regulation , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , In Vitro Techniques , MicroRNAs , Genetics , Metabolism , Protein Serine-Threonine Kinases , Genetics , Metabolism , Proto-Oncogene Proteins , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sincalide , Metabolism , TransfectionABSTRACT
Objective: To explore the effect of recombinant human interleukin-17AI (IL-17A) on the growth of colon cancer cells, and to investigate the related mechanism. Methods: The colon cancer SW480 cells were divided into control group and experimental group. The SW480 cells in control group were untreated and the SW480 cells in experimental group were added with 50 fig • L_1IL-17A. The proliferation abilities of SW480 cells in two groups were detected by CKK-8 method. The levels of IL-17A in the SW480 cells in two groups were detected by ELISA, and the expression levels of signal transducers and activators of transcription 3 (STAT3) and p-signal transducers and activators of transcriptions 3 (p-STAT3) were examined by Western blotting methed. Results: Compared with control group, the proliferation ability of the SW480 cells in experimental group was increased (P < 0 . 05). The level of IL-17A in the SW480 cells in experimental group was significantly higher than that in control group (P< 0. 01). Compared with control group, the expression levels of STAT3 and p-STAT3 proteins in the SW480 cells in experimental group were significantly higher than those in control group (P < 0 . 01). Conclusion: Recombinant protein IL-17A can stimulate the growth of colon cancer SW480 cells, which may be related to the activation of STAT3 signaling pathway.
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Objective To explore the effect of lncRNA of growth arrest-specific 5 (lncRNA GAS5) on the radiosensitivity of colon cancer cells by targeting miR-223.Methods The expressions of lncRNA GAS5 in a few of colon cancer cell lines were detected by real-time quantitative PCR (qPCR).The cell lines with low expression level of lncRNA GAS5 were selected for subsequent study.The effect of overexpression of lncRNA GAS5 on the radiosensitivity of colon cancer SW480 cells was detected by cell cloning experiments.The target gene miR-223 of lncRNA GAS5 was predicted and validated by the bioinformatics database starBase and dual luciferase reporter assays.qPCR was used to detect the expression of miR-223 in various colon cancer cell lines and the influence of lncRNA GAS5 overexpression on the expression of miR-223 in SW480 cells.Results Compared with normal human colonic epithelial cells (NCM460),the expressions of lncRNA GAS5 in the colon cancer SW480,LOVO,HT-29 and SW620 cell lines were significantly lower(t =15.25,8.69,14.42,11.62,P ＜ 0.05),with the lowest level in SW480 cells.Both overexpression of lncRNA GAS5 and down-regulation of miR-223 significantly increased the radiosensitivity of colon cancer cells by decreasing cell survival fraction (at 8 Gy,lncRNA GAS5,t =13.51,P ＜ 0.05;anti-miR-223,t =14.93,P ＜ 0.05)and promoting apoptosis (lncRNA GAS5,t =8.30,P ＜ 0.05;anti-miR-223,t =7.32,P ＜ 0.05).Bioinformatics analysis showed that the 3'sequence of lncRNA GAS5 contained the binding sites with miR-223.After overexpression or downregulation of lncRNA GAS5,the expression of miR-223 was enhanced or reduced.Conclusions The lncRNA GAS5 promotes the apoptosis of colon cancer cells and inhibits its survival by targeting miR-223 expression,thereby increases the radiosensitivity of colon cancer cells.
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Objective To explore the effect of silencing Rev1 gene on the proliferation and apoptosis induction of human colon cancer cell line THC8307 after X-ray irradiation.Methods Rev1 siRNA was transferred into THC8307 cells and its transferring efficiency was tested by RT-PCR and Western blot assay.The cells were divided into blank control group,negative control group and radiation group.After 6 Gy irradiation,cell proliferation were detected by MTI assay,cell apoptosis were detected by flow cytometry (FCM),and the expressions of PCNA,γ-H2AX,P53,Bax,and Bcl-2 proteins in each group were detected by Western blot.Results Compared with the blank control group,after 6 Gy irradiation,the proliferation rate of the silenced Rev1 was reduced (t =7.53,P ＜ 0.05) and apoptosis rate was increased (t =6.23,P ＜ 0.05).The expression of PCNA and Bcl-2 decreased (t =4.39,6.13 P ＜0.05),and the expressions of γ-H2AX,P53 and Bax increased (t =5.48,5.09,3.32,P ＜ 0.05).Conclusions Silencing Rev1 gene inhibits the proliferation of colon cancer cells,promotes apoptosis,and increases cell radiosensitivity to X-rays.
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Objective: To investigate the anti-colon cancer effects of ethylacetate fraction from Orostachys japonicus (O. japonicus) on HT-29 cancer cells. Methods: The viability of HT-29 cells was assayed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method. Apoptosis induction and cell cycle inhibition were confirmed by fluorescein isothiocyanate and propidium iodide staining using flow cytometry. Morphological changes in the nucleus were observed, using a fluorescence microscope with 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. The expression levels of the upstream and downstream proteins involved in the anti-cancer mechanism were confirmed by Western blotting. Results: After treating HT-29 cells with different concentrations of ethylacetate fraction from O. japonicus, the viability of cells decreased in a concentration-dependent manner, while apoptosis induction and apoptotic body formation increased. Cell cycle analysis showed that the arrest occurred at the sub-G
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Objective:To investigate the anti-colon cancer effects of ethylacetate fraction from Orostachys japonicus (O. japonicus) on HT-29 cancer cells.Methods:The viability of HT-29 cells was assayed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method. Apoptosis induction and cell cycle inhibition were confirmed by fluorescein isothiocyanate and propidium iodide staining using flow cytometry. Morphological changes in the nucleus were observed, using a fluorescence microscope with 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. The expression levels of the upstream and downstream proteins involved in the anti-cancer mechanism were confirmed by Western blotting.Results:After treating HT-29 cells with different concentrations of ethylacetate fraction from O. japonicus, the viability of cells decreased in a concentration-dependent manner, while apoptosis induction and apoptotic body formation increased. Cell cycle analysis showed that the arrest occurred at the sub-GConclusions:Combining the above results, it is thought that the survival of HT-29 cells is suppressed by ethylacetate fraction from O. japonicus through mitochondrial regulation-induced caspase cascade activation, induction of apoptosis and cell cycle arrest.
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Objective To study the effect of quercetin on the apoptosis of human colon cancer HT-29 cells and observe the possible mechanism.Methods Human colon cancer HT-29 cells were cultured in vitro and divided into four groups, including the control group, the 16μg/ml quercetin group, the Traf6 inhibitor group and the 16μg/ml quercetin + inhibitor group. The cells in control group were cultured with complete medium and other groups were treated with quercetin or/and Traf6 inhibitor. Flow cytometry was used to observe the impact of quercetin on the apoptosis of HT-29 cells; Western Blot technology was used to detect the expression levels of Traf6, TAK1, p-TAK1, caspase-3, Bax and Bcl-2; RT-PCR was used to investigate the expression level of Traf6 mRNA after treating for 24 h.Results Compared with the 16μg/ml quercetin group, the expression levels of Traf6 (0.59 ± 0.03 vs. 0.96 ± 0.04), p-TAK1 (0.43 ± 0.02vs. 0.72 ± 0.04), caspase-3 (0.59 ± 0.03vs. 0.70 ± 0.04), and Bax (0.48 ± 0.03vs.0.67 ± 0.04) were significantly decreased in 16μg/ml quercetin + inhibitor group(P<0.05). while the expression levels of Bcl-2 (0.54 ± 0.03vs. 0.44 ± 0.02) was significantly increased (P<0.05).Conclusions Quercetin can induce the apoptosis of human colon cancer HT-29 cells and the effective mechanism may relate to the activation of Traf6/TAK1 signaling pathway.
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Objective To study the effect of quercetin on the apoptosis of human colon cancer HT-29 cells and observe the possible mechanism.Methods Human colon cancer HT-29 cells were cultured in vitro and divided into four groups, including the control group, the 16μg/ml quercetin group, the Traf6 inhibitor group and the 16μg/ml quercetin + inhibitor group. The cells in control group were cultured with complete medium and other groups were treated with quercetin or/and Traf6 inhibitor. Flow cytometry was used to observe the impact of quercetin on the apoptosis of HT-29 cells; Western Blot technology was used to detect the expression levels of Traf6, TAK1, p-TAK1, caspase-3, Bax and Bcl-2; RT-PCR was used to investigate the expression level of Traf6 mRNA after treating for 24 h.Results Compared with the 16μg/ml quercetin group, the expression levels of Traf6 (0.59 ± 0.03 vs. 0.96 ± 0.04), p-TAK1 (0.43 ± 0.02vs. 0.72 ± 0.04), caspase-3 (0.59 ± 0.03vs. 0.70 ± 0.04), and Bax (0.48 ± 0.03vs.0.67 ± 0.04) were significantly decreased in 16μg/ml quercetin + inhibitor group(P<0.05). while the expression levels of Bcl-2 (0.54 ± 0.03vs. 0.44 ± 0.02) was significantly increased (P<0.05).Conclusions Quercetin can induce the apoptosis of human colon cancer HT-29 cells and the effective mechanism may relate to the activation of Traf6/TAK1 signaling pathway.
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Aim To investigate the effects of dalbinol on proliferation and apoptosis of human colon cancer HCT1 16 cells and its mechanisms.Methods Anti-proliferative effect of dalbinol was evaluated by MTT assay.The morphological changes of apoptosis were observed by Hoechst33342 staining.Apoptotic rate and ROS generation were analyzed by flow cytometry.The related proteins of Wnt/β-catenin pathway and the ap-optosis-associated proteins expression were measured by Western blot.Results The growth of HCT1 16 treated with dalbinol was inhibited in a dose and time dependent manner with IC50 (4.8 ±0.53 ),(2.5 ± 0.43)and (0.6 ±0.22)μmol·L-1 at 24,48 and 72 h,respectively.Typical morphological changes of ap-optosis such as cell shrinkage,karyopyknosis and nu-clear condensation were observed by Hoechst33342 staining.Meanwhile,the apoptotic rate and intracellu-lar ROS generation of dalbinol were both increased dose-dependently. Western blot results showed that dalbinol could activate the expression of cleaved Caspase-3 and cleaved PARP by decreasing anti-apop-totic protein levels such as Bcl-2 and Mcl-1 and in-creasing pro-apoptotic protein levels such as Bax and Bim,which induced further apoptosis.Moreover,dal-binol can reduce the protein expression of the total and nuclear β-catenin,but not cytoplasmic β-catenin by suppressing the protein expression of Dvl-2 and GSK-3β(pS9 ),as well as its target proteins c-Myc and Sur-vivin.Conclusion dalbinol can induce apoptosis in colon cancer HCT1 16 cells by upregulating the intra-cellular ROS generation and suppressing Dvl/GSK-3β/β-catenin pathway.
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[ ABSTRACT] AIM:To investigate the effects of sphingosine kinase l ( SphK1) and focal adhesion kinase ( FAK) on the epithelial-mesenchymal transition ( EMT) of human colon cancer HCT 116 cells.METHODS:Human colon cancer HCT116 cells were divided into 3 groups.N, N-dimethylsphingosine (DMS) was used to suppress the activity of SphK1. PF573228 was used to suppress the activation of FAK .The cells treated with equal volume of culture medium severed as control group.The cell viability was measured by MTT assay .The protein expression of SphK1, FAK and the EMT relative protein E-cadherin, N-cadherin, vimentin and matrix metalloproteinase (MMP) 2 was analyzed by Western blot.The mR-NA expression of SphK1, sphingosine-1-phosphate (S1P), FAK, E-cadherin and vimentin was detected by real-time PCR. The ability of tumor cell migration was measured by wound-healing assay.RESULTS:The cell viability of HCT116 cells was suppressed by DMS and PF 573228 in dose and time dependent manners .DMS significantly suppressed the expression of SphK1, FAK, N-cadherin, vimentin and MMP2, meanwhile enhanced the expression of E-cadherin.PF573228 reduced the expression of FAK , SphK1, N-cadherin, vimentin and MMP2, meanwhile increased the expression of E-cadherin (P<0.01).In addition, the migration ability of HCT116 cells was significantly decreased by treating with DMS and PF573228 (P<0.01).Compared with control group , the mRNA expression of FAK, SphK1, S1P and vimentin was de-creased, while the expression of E-cadherin was increased significantly in PF573228 group and DMS group (P<0.05). CONCLUSION:SphK1 and FAK signaling pathways may play an important role in the occurrence of EMT in the colon cancer HCT116 cells.
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Objective:To explore the regulatory effects of proliferation and apoptosis on THC-8307 by MMS2 siRNA and P53 siRNA.Methods:We experimentally suppressed the MMS2 and P53 expression in human colon cancer cells by the interference RNA technology ( RNAi) as monitored by Real-time qRT-PCR and Western blot.THC-8307 cells that express rate significantly reduced were collected as case group , while using untreated cells as the blank control group , and mock-treated cells as the negative control group.After separately interfering the target genes in each group ,test the relationship and expression level of the two genes.Utilizing flow cytometry techniques to test cells proliferation and apoptosis rate of each group.Results: Compared to the control group , colon cancer cells in which MMS2 and P53 were silenced displayed significant increase of P53,MMS2 mRNA and protein levels(P<0.05).MMS2-depleted cells displayed increase in apoptosis rates ,for both early and later stages ( P<0.05 ).Conclusion: MMS2 and P53 negatively regulate each other in colon cancer cells proliferation and apoptosis .
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Aim To investigate the promoting apoptosis effect of artesunate( ART) on human colon cancer Lovo cells and its mechanisms. Methods MTT assay was performed to determine the anti-proliferative effect of artesunate. Flow cytometry assay and electron micros-copy( EM) were used to evaluate the apoptotic effect of artesunate. Luciferase reporter assay was introduced to measure the activation of Wnt/β-catenin pathway. Western blot was used to detect the pathway-related protein levels of β-catenin, GSK-3β,c-Myc and apop-tosis-related protein level of casepase-3 . Results Compared with the control group, the inhibitory rate of cell proliferation at 72 h and 320 μmol·L-1 ART was (78. 99 ± 1. 95 )% ( F =898. 301, P =0. 000 ); the cell apoptotic rate at 24 h and 160 μmol · L-1 ART was(19. 00 ± 0. 05)% and morphological signs of cell apoptosis were found by EM;the transcriptional activi-ty of TCF4/LEF at 24 h and 160 μmol·L-1 ART was (0. 36 ± 0. 30)%(F =470. 954,P <0. 01); the ex-pressions of caspase-3 and GSK-3β were significantly increased, whileβ-catenin and c-Myc were significant-ly decreased when treated with different concentrations of ART for 48 h ( P <0. 01 ) . Conclusion ART may significantly inhibit proliferation and promote apoptosis of Lovo cells probably by inactivating Wnt/β-catenin pathway.
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Objective To establish model of the chicken embryo transplantation of human colon cancer cells ,and investigate the effect of Solanine、VEGF antibody and Solanine combined with VEGF antibody on human colon cancer cells induce tumor angio‐genesis and tumor proliferation .Methods The model of the chicken embryo transplantation of human colon cancer HT‐29 cells were divided into three experimental group and control group .We added to the chick embryo chorioallantoic membrane with Sola‐nine、VEGF antibody and Solanine+ VEGF antibody mixture ,PBS was added to the control group .Then we analysed picture through the stereomicroscope and IPP 6 .0 image analysis software ,using immunohistochemistry envision method to detect of CD34 antigen and ki‐67 antigen ,and observing effect of Solanine group ,VEGF antibody group ,Solanine+ VEGF antibody group and the effect on the tumor angiogenesis and tumor proliferation .Results The tumor angiogenesis ,CD34 antigen and ki‐67 antigen of Sola‐nine+VEGF antibody group were significantly better than those of VEGF antibody group and Solanine group(P<0 .01);VEGF antibody group had statistical significant difference with Solanine group(P<0 .01);the effect of other three groups were better than that of the control group(P<0 .01) .Conclusion Solanine、VEGF antibody and Solanine combined with VEGF antibody could in‐hibit tumor angiogenesis and tumor proliferation of human colon cancer cell line HT‐29 to induce .It provides a new way for anti‐an‐giogenes .
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OBJECTIVE To observe the regulation effect of chidamide on energy metabolism in HCT-8 and HT-29 cells. METHODS HCT-8 and HT-29 cells were treated with chidamide 5,10 and 20 μmol · L-1. Morphological changes of these cells were observed under an ordinary optical microscope. Cell proliferation was detected by MTT. ATP production was determined by CellTiter-Glo? assay kit. Metabolic changes were tested by glycolytic stress kit. The mRNA level of lactate dehydrogenase A (LDH-A)was analyzed by real-time quantitative PCR,whereas the protein level of LDH-A was analyzed by Western blotting. RESULTS Compared with control group,cell morphology of HCT-8 and HT-29 cells in chidamide treated group was irregular,accompanied by deformation,shrinkage and cell debris, and the inhibitory rate of proliferation increased(P<0.05). There was no significant difference in ATP total content between chidamide 5 and 10 μmol · L-1 16 h treatment groups,but in chidamide 20 μmol · L-1 treatment group it was decreased(P<0.05). Chidamide 20μmol · L-1 had no effect on oxygen consumption rate, but glycolysis ATP generation rate was reduced by 30.7% and 37.9%(P<0.05),respectively. Chidamide 20μmol · L-1 had no effect on LDH-A mRNA level,but it decreased the protein level of LDH-A(P<0.01). CONCLUSION Chidamide can abate the respiratory metabolic ability of HCT-8 and HT-29 cells. The mechanism may be related to the down-regulation of LDH-A.
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Objective To study PPARγ inhibitor(GW9662) ,on colon cancer SW480 cell proliferation and apoptosis intervened by Nimesulide(N) in vitro ,in order to investigate the role of PPARγpathway in colon cancer cell proliferation inhibition and apop‐tosis promotion induced by Nimesulide .Methods Cells were divided into 4 groups ,namely :the control group ,GW9662 group (GW9662 0 .1 ,0 .5 ,1 .0 ,5 .0μmol/L) ,N group ,GW9662+N group .MTT assay and FCM were used to determine proliferation ,ap‐optosis and cell cycle of SW480 cells .And the expression of PPARγ,p21Waf1 ,p27Kip1 ,Bcl‐2 ,Bax ,VEGF proteins were measured by Western‐blot .Results N inhibited SW480 cells proliferation in a time‐dependent manner (P0 .05) . Cell apoptosis rate of group N increased significantly ,compared with control group(P<0 .01) .The apoptosis rates of SW480 cells incubated with Nimesulide and GW9662 dropped significantly compared with Nimesulide alone (P<0 .01) .Above results showed that GW9662 could attenuate the effect of nimesulide on cell apoptosis and cell cycle .The results of Western‐blot :Compared with the control group ,the expression of PPARγ,p21Waf1 ,p27Kip1 ,Bax protein were up‐regulated significantly in nimesulide group(P<0 .05 or P<0 .01) ,but Bcl‐2 and VEGF were down‐regulated significantly(P<0 .01) .Compared with the nimesulide group ,the expres‐sion of PPARγ,p21Waf1 , p27Kip1 and Bax protein were down‐regulated obviously in GW9662+N group(P<0 .05 or P<0 .01) .Corre‐spondingly ,Bcl‐2 and VEGF were up‐regulated obviously(P<0 .05) .Conclusion N could effectively inhibit SW480 cell prolifera‐tion and induce its apoptosis .PPARγpathway may play an important role in proliferation inhibition and apoptosis induced by Nime‐sulide in colon cancer cell .
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Objective To investigate the effect of emodin on immune suppression function of regulatory T cells in a mouse model of CT26 colon cancer. Methods Twenty-four mice were divided into the negative control group, the emodin group and the tumor group. The populations of CD8+CD3+T cells, the T cells producing IFN-γand the CD4+CD25+Tregs secreting IL-10 in different mouse tissues were detected by flow cytometry. Levels of IFN-γ, TNF- β1 and IL-10 in serum were determined by ELISA. Results Emodin could significantly increase the percent of CD8+CD3+T cells in tumor (P < 0.05) and improve the ability of IFN-γ secretion in T cells from peripheral blood and lymph nodes (P < 0.05). Emodin could reduce the levels of IFN-γ, TNF-β1 and IL-10 in the serum (P < 0.01) and inhibit IL-10 secretion in CD4+CD25+ Tregs (P < 0.01). Conclusion Emodin possesses the antitumor effect by affecting the immunosuppressive function of Tregs cells.
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Objective:To isolate and identify the anticancer compound against proliferation of human colon cancer cells from ethyl acetate (EtOAc) extract of Phellinus linteus grown on germinated brown rice (PB). Methods: EtOAc extract of PB was partitioned with n-hexane, EtOAc, and water-saturated n-butanol. Anticancer compound of n-hexane layer was isolated and identified by HPLC and NMR, respectively. Cytotoxicity against HT-29 cells was tested by SRB assay. Results: The n-hexane layer obtained after solvent fractionation of PB EtOAc extracts showed a potent anticancer activity against the HT-29 cell line. Atractylenolide I, a eudesmane-type sesquiterpene lactone, a major anticancer substance of PB, was isolated from the n-hexane layer by silica gel column chromatography and preparative-HPLC. This structure was elucidated by one-and two-dimensional NMR spectroscopic data. Atractylenolide I has not been reported in mushrooms or rice as of yet. The isolated compound dose-dependently inhibited the growth of HT-29 human colon cancer cells. Conclusions:Atractylenolide I might contribute to the anticancer effect of PB.
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The aim of the study was to investigate the inhibitory effects of calcium against intestinal cancer in vitro and in vivo. We first investigated the effects of calcium treatment in HCT116 and HT29 human colon cancer cells. At the concentration range of 0.8-2.4 mM, calcium significantly inhibited cell growth (by 9-29%), attachment (by 12-26%), invasion (by 15-31%), and migration (by 19-61%). An immunofluorescence microscope analysis showed that the treatment with calcium (1.6 mM) for 24 h increased plasma membrane beta-catenin but decreased nuclear beta-catenin levels in HT29 cells. We then investigated the effect of dietary calcium on intestinal tumorigenesis in ApcMin/+ mice. Mice received dietary treatment starting at 6 weeks of age for the consecutive 8 weeks. The basal control diet contained high-fat (20% mixed lipids by weight) and low-calcium (1.4 mg/g diet) to mimic the average Western diet, while the treatment diet contained an enriched level of calcium (5.2 mg calcium/g diet). The dietary calcium treatment decreased the total number of small intestinal tumors (by 31.4%; P or = 2 mm in diameter, showing a 75.6% inhibition in the small intestinal tumor multiplicity (P < 0.001). Immunohistochemical analysis showed significantly reduced nuclear staining of beta-catenin (expressed as nuclear positivity), but increased plasma membrane staining of beta-catenin, in the adenomas from the calcium-treated groups in comparison to those from the control group (P < 0.001). These results demonstrate intestinal cancer inhibitory effects of calcium both in human colon cancer cells and Apc Min/+ mice. The decreased beta-catenin nuclear localization caused by the calcium treatment may contribute to the inhibitory action.