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Objective:To investigate the effects of capsaicin on colon cancer SW480 cells and the underlying molecular mechanism through the transient receptor potential vanilloid 1（TRPV1）. Method:Capsaicin groups with different concentrations and a blank group were set up. The cell viability was detected by cell counting kit-8 （CCK-8） after SW480 cells were treated with capsaicin（50，100，200，300，400，500，600，800，1 000 μmol·L<sup>-1</sup>） for 12，24，and 48 h to select the concentration of capsaicin which can effectively inhibit proliferation. The cell cycle and apoptosis were detected by flow cytometry after SW480 cells were treated with capsaicin （200，400，800 μmol·L<sup>-1</sup>） for 24 h. The protein expression levels of TRPV1，p53，p-p53，B-cell lymphoma-2（Bcl-2），Bcl-2-associated X protein（Bax），cleaved cysteinyl aspartate-specific protease-3（cleaved Caspase-3），cleaved Caspase-8，and cleaved poly adenosine diphosphate ribose polymerase （PARP） were detected by Western blot after SW480 cells were treated with capsaicin （200，400 μmol·L<sup>-1</sup>） for 24 h．In addition，the apoptosis was detected after SW480 cells were treated with TRPV1 microRNA（mRNA） and capsaicin（200 μmol·L<sup>-1</sup>）. Western blot analysis was used to detect the protein expression levels of the above proteins. Result:As compared with the blank group，capsaicin（≥200 μmol·L<sup>-1</sup>）significantly inhibited the cell viability of SW480 cells（<italic>P</italic><0.01） in dose- and time-dependent manners. The cell cycle was arrested in G<sub>2</sub>/M phase by 200 and 400 μmol·L<sup>-1</sup> capsaicin treatment，and arrested in G<sub>1</sub> phase by 800 μmol·L<sup>-1</sup> capsaicin treatment （<italic>P</italic><0.05）. Flow cytometry showed that capsaicin （200， 400， 800 μmol·L<sup>-1</sup>） significantly promoted apoptosis of SW480 cells simultaneously（<italic>P</italic><0.05，<italic>P</italic><0.01）. Western blot showed that capsaicin （200，400 μmol·L<sup>-1</sup>） significantly up-regulated the protein levels of apoptosis-related proteins（p53，p-p53，Bax，cleaved Caspase-3，cleaved Caspase-8，and cleaved PARP） （<italic>P</italic><0.05，<italic>P</italic><0.01），and significantly down-regulated Bcl-2（<italic>P</italic><0.01）. In addition，siRNA-mediated knockdown of TRPV1 significantly attenuated capsaicin-induced apoptosis and the protein levels of apoptosis-related proteins in SW480 cells（<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:Capsaicin can inhibit cell proliferation，arrest cell cycle，and induce apoptosis of SW480 cells，and the possible mechanism may be related to TRPV1 activation.
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Objective@#To analyze the effects of Clostridium difficile toxin B (TcdB) on the proliferation and apoptosis of colon cancer cell line SW480 and the possible mechanisms related to cell apoptosis.@*Methods@#SW480 cells were treated with different concentrations of TcdB. Cell proliferation was detected by MTT assay. Cell apoptosis and mitochondrial membrane potential were measured with flow cytometry.@*Results@#TcdB significantly inhibited the proliferation of SW480 cells in a time-concentration dependent manner and the inhibition rate reached 46.36% at 48 h. Flow cytometry results showed that TcdB could induce the apoptosis of SW480 cells in a time-concentration dependent manner and a 20.83% apoptosis rate was induced by 800 ng/ml of TcdB at 48 h.@*Conclusions@#TcdB could inhibit the proliferation and induce the apoptosis of colon cancer SW480 cells, and the possible mechanisms might be relate to the initiation of mitochondrial apoptosis pathway.
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Objective: To study the effects of ginsenoside CK on proliferation and apoptosis of human colon cancer cell line SW480, and further explore the mechanism. Methods: Cell viability was measured by CCK-8 assay. Cell cycle, apoptosis, reactive oxygen species (ROS) levels and changes in mitochondrial membrane potential were measured by flow cytometry. Hoechst staining further detected apoptosis. Western blotting was used to detect the release of cytochrome C and the expression of apoptosis-related proteins such as Bcl-2, Bax and cleaved Caspase-3. Results: Ginsenoside CK had a significant inhibitory effect on the proliferation of human colon cancer cell line SW480. Ginsenoside CK induced SW480 cells arrest in G0/G1 phase, promoted early apoptotic cells, significantly increased intracellular ROS levels and reduced the MMP level. Ginsenoside CK promoted the expression of Bax and cleaved-Caspase-3 and inhibited the expression of Bcl-2. In addition, ginsenoside CK released a large amount of cytochrome C in SW480 cells. Conclusion: Ginsenoside CK has a significant inhibitory effect on the proliferation of human colon cancer cell line SW480. The mechanism may be through the promotion of mitochondrial superoxide elevation, resulting in a significant increase in intracellular ROS levels and a significant decrease in MMP level, further leading to the release of cytochrome C, the up-regulated expression of Bax, the down-regulated expression of Bcl-2, and ultimately leading to apoptosis of cells.
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Objective To analyze the effects of Clostridium difficile toxin B (TcdB) on the prolif-eration and apoptosis of colon cancer cell line SW480 and the possible mechanisms related to cell apoptosis. Methods SW480 cells were treated with different concentrations of TcdB. Cell proliferation was detected by MTT assay. Cell apoptosis and mitochondrial membrane potential were measured with flow cytometry. Re-sults TcdB significantly inhibited the proliferation of SW480 cells in a time-concentration dependent man-ner and the inhibition rate reached 46. 36％ at 48 h. Flow cytometry results showed that TcdB could induce the apoptosis of SW480 cells in a time-concentration dependent manner and a 20. 83％ apoptosis rate was in-duced by 800 ng/ml of TcdB at48 h. Conclusions TcdB could inhibit the proliferation and induce the ap-optosis of colon cancer SW480 cells, and the possible mechanisms might be relate to the initiation of mito-chondrial apoptosis pathway.
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Background: MicroRNAs are small noncoding RNAs which modulate gene expression at different levels. It has been shown that downregulation of miR-34a occurs in varieties of cancers including colorectal cancer (CRC). In this study, we investigated the potential tumor inhibitory effects of miR-34a alone or in combination with paclitaxel in CRC cells. Materials and Methods: SW480 cells were transduced with lentiviral overexpressed miR-34a. First, using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, the effect of miR-34a induction alone or in combination with paclitaxel on the cell viability and cell proliferation were estimated. Then, the expression level of target genes was measured using quantitative reverse transcription-polymerase chain reaction analysis. Eventually, the role of miR-34a and paclitaxel on cell cycle were determined with flow cytometry. Results: Gene expression analysis showed that miR-34a downregulates the expression of BCL2 and SIRT1 genes at mRNA level. Furthermore, miR-34a has a potential to reduce cell viability and cell cycle arrest at G1 phase. Combination of paclitaxel with overexpression of miR-34a significantly decreased cell viability compared to cell treated with miR-34a or paclitaxel alone. Interestingly, a combination of miR-34a and paclitaxel arrested cell cycle at two phases. Conclusion: Our results suggested that combination therapy of miR-34a and paclitaxel could be considered as the potential treatment of CRC.
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AIM: To explore the effect of pinobanksin-3-acetate (PB3A) on microRNA (miRNA) expression profile of human colon cancer cells for providing new methods of treatment of colon cancer and development of targeted drug.METHODS: The method of miRNA expression profiling was used to observe the miRNA differential expression in human colon cancer SW480 cells after treated with PB3A.The expression of miRNA-198 and miRNA-296-5p in the SW480 cells was detected by RT-qPCR.The network databases of miRWalk, MicroT, miRanda and so on were used to predict the target genes regulated by these miRNAs, and pathway significant enrichment analysis was performed.RESULTS: miRNA microarray analysis showed that after treated with propolis flavonoid PB3A for 24 h, 267 miRNAs with differential expression twice or more in the SW480 cells were observed.Among them, there were 30 miRNAs with 10-fold or more differential expression, in which 28 were up-regulated and 2 were down-regulated.The results of RT-qPCR showed that the expression levels of miRNA-198 and miRNA-296-5p were consistent with the results of miRNA microarray analysis, and the difference was statistically significant (P<0.05).Bioinformatic analysis revealed that miRNA-198 has 859 target genes, and miRNA-296-5p has 906 target genes.The target genes of miRNA-198 were clustered in pathways in cancer, axon guidance, Wnt signaling pathway, regulation of actin cytoskeleton, insulin signaling pathway and MAPK signaling pathway, while the target genes of miRNA-296-5p were clustered in axon guidance, Wnt signaling pathway, MAPK signaling pathway, endocytosis, melanogenesis, insulin signaling pathway and calcium signaling pathway.CONCLUSION: Propolis flavonoid PB3A affects the expression of miRNA in colon cancer SW480 cells.The abnormal expression of miRNA-198 and miRNA-296-5p may be involved in the inhibitory effect of PB3A on colon cancer.
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Objective:To investigate the promotion effect of 4,5,6,7-tetrabromobenzotriazole (TBB)on the apoptosis of human colon cancer SW480 cells,and to explore its possible mechanism.Methods:The human colon cancer SW480 cells at logarithmic growth phase were divided into control group (0 μmol·L-1 TBB)and experiment group (1,3,10,30,and 100 μmol·L-1 TBB).The viability of cells was measured by MTT assay;the apoptotic rate of the SW480 cells and the level of intracellular reactive oxygen species (ROS)were analyzed by Annexin Ⅴ-FITC/PI double staining and flow cytometry.The expression levels of anti-apoptotic proteins p-Akt and Bcl-2,and pro-apoptotic proteins Bad, pro-caspase-9 and cleaved-caspase-3 were detected by Western blotting method. Results:The MTT results showed that the viabilities of SW480 cells in experiment group were decreased in a dose-dependent manner,which were lower than that in control group (P < 0.05).The Annexin Ⅴ-FITC/PI double staining and flow cytometry results showed that the apoptotic rates of SW480 cells in experiment group (3,6,12, and 24 h)were significantly higher than that in control group (0 h)(P <0.05).The flow cytometry results showed the levels of ROS in SW480 cells after treated with TBB for 3,6,12 and 24 h were higher than that in 0 h group (P <0.05).The Western blotting results showed that the expression levels of anti-apoptotic proteins p-Akt and Bcl-2 in SW480 cells in experiment group (3,6,12,and 24 h)were decreased obviously,whereas the expression levels of the pro-apoptotic proteins Bad and cleaved-caspase-3 were increased and the expression level of pro-caspase-9 was decreased compared with those in control group (0 h) (P < 0.05 ).Conclusion: TBB could inhibit the cell proliferation and induce the apoptosis of human colon cancer SW480 cells,and its mechanism may be related to the inhibition of the activity of Akt and the promotion of the level of intracellular ROS.
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Objective To investigate the effect of ampelopsin on apoptosis induction and cell growth inhibition in Human Colon cancer SW480 cells in vitro.Methods Treated with ampelopsin at several concentrations, MTT and flow cytometry was used to detect the inhibition rate and apoptotic rate of SW480 cells.Western-blot was used to investigate expression of Bcl-2 family pro-tein.Results Significant difference of cell growth inhibition rate was observed among all groups after treated with ampelopsin ( p0.05).The similar results were observed in the experiment on apoptosis induction.Level of Bcl-xL was significantly up-regulated.Level of Bax, Bid, Caspase-3, p-Ca-pase-9 and Caspase-9 was significantly down-regulated after treatment of ampelopsin.Conclusion Ampelopsin can inhibit cell growth and induce apoptosis of SW480.Bcl-2 family protein might be involved in the progress.
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Objective To investigate the effect of eicosapentaenoic acid(EPA)on the apoptosis of human colon cancer SW480 cells and the mechanisms.Methods Mitochondrial membrane potential was detected,the quan-tity of cytochrome C was analyzed by Elisa kit,and the expression of cleaved caspase -9 and caspase -3 was detected by Western Blot.Results After treatment with EPA (0μg/mL,42.1μg/mL,84.2μg/mL,168.4μg/mL),the mitochondrial membrane potential(Δψm)of SW480 cells were declined (P <0.05 ),the values were (99.71 ± 0.04)%,(95.04 ±0.10)%,(88.65 ±0.41)% and (73.60 ±1.20)%(t =5.161,6.302,4.601,5.198,all P <0.05).The quantity of cytochrome C in cytosol was increased significantly compared with no treatment group,and the values were (12.8 ±1.2)ng/mL,(115.5 ±3.5)ng/mL,(290.5 ±5.2)ng/mL and (262.0 ±12.5 )ng/mL in different EPA treatment groups(t =6.345,6.013,5.846,4.613,all P <0.01).The expression of cleaved caspase -9 and caspase -3 were significantly increased.Conclusion EPA inhibits SW480 cells growth and induces apoptosis in a dose dependent manner.This action may be mediated by mitochondria -mediated intrinsic apoptosis pathway,cyto-chrome C release,and caspase -9 and caspase -3 activation.
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Objective To investigate the influence of baicalin in human colon cancer SW480 cells,and to clarify its mechanism.Methods The SW480 cells were cultured and divided into blank control and 25,50 and 100 μmol·L-1 baicalin groups.The proliferation activity was detected with CCK-8 assay.The morphological changes of SW480 cells were detected by Annexin Ⅴ-FITC and DAPI coloration.The protein expression levels of Bcl-2,caspase 3 and caspase 9 were detected by Western blotting method. Results The CCK-8 assay results showed that the proliferation activities of SW480 cells in 25,50 and 100 μmol· L-1 baicalin groups were decreased significantly compared with blank control group at the time points of 24 h,48 h and 72 h (P <0.01),the proliferation activities of SW480 cells in 25 μmol·L-1 baicalin groups were decreased significantly compared with blank control group at the time points of 48 and 72 h (P <0.01).Cell shrinkage and nucleus fragmentation were observed in the SW480 cells after treated with 50 μmol·L-1 baicalin for 48 h.The Western blotting assay results showed that compared with blank control group,the protein expression levels of caspase 3 and caspase 9 in 25,50 and 100 μmol· L-1 baicalin groups were increased significantly (P <0.05 or P <0.01),and the protein expression levels of Bcl 2 in 25, 50 and 100 μmol·L-1 baicalin groups were decreased significantly (P <0.05 or P <0.01).Conclusion Baicalin can induce the apoptosis in SW480 cells,and the effect might be involved with the mitochondrial apoptotic pathway.
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Objective:This work aims to investigate diallyl disulfide (DADS) inhibition of cell migration and invasion in human colon cancer SW480 cells through the Rac1-ADF/cofilin1 pathway. Methods:The potential of cell migration and invasion was examined by scratch healing assay and transwell membrane assay. The expression of Rac1-ADF/cofilin1 pathway was detected by RT-PCR and Western blot. Results:After the SW480 cells were treated with 40 and 50 mg·L-1 of DADS for 24 h, the number of transmembrane cells through the Matrigel obviously decreased by 57.12%and 64.59%, respectively (P0.05). After the treatment with 45 mg·L-1 of DADS for 6, 12, 24, and 48 h, the expression of Rac1, Rock1, PAK1, LIMK1, and Destrin proteins respectively decreased in a time-dependent manner compared with the control group (P0.05). Moreover, the expression of p-LIMK1 and p-cofilin1 notably decreased in a time-dependent manner (P<0.05). Conclusion:DADS inhibits cell migration and invasion, which is related to the down-regulation of Rac1, Rock1, PAK1, LIMK1, p-LIMK1, p-cofilin1, and destrin through the Rac1-ADF/cofilin1 pathway.
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Objective To evaluate the proliferation and invasion potential of human colon cancer SW480 cells transfected by AP-2α gene in vitro. Methods pcDNA3.1 (+)-AP-2α was created by cloning AP-2α eDNA into the EcoRI site of poDNA3.1 (+). LipofectamineTM 2000 Reagent was used to mediate the transfection of pcDNA3.1(+)-AP-2α and pcDNA3.1 (+), and normal SW480 celia were cultured as a negative control. The mRNA and protein level of AP-2α in the cells of each group were detected 48 h after being transfected with plasmids above by RT-PCR and Western blotting analysis. The cell proliferation and invasion potential were examined by colony formation assay and Transwell invasion assay respectively. Results Lack of AP-2α protein expression in SW480 cells was verified by western blot analysis. After being transfected with AP-2α gene, the mRNA amount and protein expression increased dramatically, while the colony formation efficiency decreased(P <0.05), the cell proliferation in soft agar was inhibited, and the ability of its invasion dropped off(P <0.05) in vitro. Conclusion AP-2α gene suppresses the proliferation and invasion potential of human colon cancer SW480 cells in vitro.
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Objective To investigate whether prostaglandin E2(PGE2) can promote the ability of adhesion.migration and invasion of colorectal cancer cells SW480 and its mechanism.Methods Extrinsic source PGE2 and the antagonist of EP1 SC19220 were added to the culture media. MTr assay was used to identify the adhesion ability of SW480 cells. Migration ability was tested by transwell plate.The invasion ability was tested bv ECM gel coated transweU plate. RT-PCR and western blotting were used to detect the mRNA and protein level of vascular endothelial growth factor (VEGF). Results The adhesion.invasion and migration ratio of SW480 ceils were all increased significantly after treated with PGEh the A values of adhesion ceils increased from 0.207±0.009 to 0.417±0.088. Migration cells increased from 6.33±0.33 to 43.33±0.88.invasion cells increased from 3.67±0.34 to 26.33±0.89(P＜0.05).The adhesion.migration and invasion cells of PGE2+SC 19220 group decreased significantly compared to PGE2 group.The A values of adhesion cells decreased from 0.417±0.088 to 0.140±0.006. Migration cells decreased from 43.33±0.88 to 28.00±0.58.invasion cells decreased from 26.33±0.89 to 5.67±0.33 (P＜0.05).The results of RT-PCR and western blotting showed that the expression of VEGF mRNA and protein increased in a dose dependent manner after PGE2 treatment. Conclusion The ability of adhesion. Migration and invasion of SW480increased after PGE2 was added to the culture media.It may be related to the upregulation of VEGF.
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In order to examine the effect of GRIM 19 on colon cancer cell SW480, the recombinant adenovirus carrying GRIM19 gene was constructed and transfected into SW480 cells. GRIMI9 cDNA was amplified by PCR with the template pcxn2-GRlMl9 and cloned into the shuttle plasmid pAdTrack-CMV. The plasmid pAdTrack-CMV-GRIM19 was linearized by PmeI and homologously recombined with bone plasmid pAdEasy-1 in BJ5183, followed by identification by enzyme diges- tion. After transfection of linearized pAd-GRIM19 with PacI into HEK293 cells, Ad-GRIMI9 was obtained and amplified by 3 circles. SW480 cells were infected with Ad-GRIM19. The apoptosis rate was detected by flow cytometry. Agarose electrophoresis revealed the bands of recombinant plasmids identified by enzyme digestion were in the right range corresponding with expectation. Under the fluorescent microscopy, the package of Ad-GRIM19 in HEK293 cells and the expression of Ad-GRIM19 in SW480 cells were observed. The transfection of Ad-GRIM19 into SW480 cells in-creased the apoptosis rate of SW480 cells as compared with controls. It was concluded that Ad-GRIM19 was successfully constructed and the overexpression of GRIM19 in colon cancer cell lines could promote the apoptotic cell death.
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Objective To investigate the effect of transforming growth factor-?1 (TGF-?1) on cell cycle,cell proliferation,and invasive capacity of human colon cancer cells of the line SW480,for its role in colon carcinogenesis and development,as well as its application in gene therapy for colon cancer with RNA interference.Methods The recombinant expressing plasmids pEGFP-N1-TGF-?1 was constructed and transfected into SW480 cells by Lipofectamine 2000.The expression of TGF-?1 mRNA and protein in the transfected SW480 cells were detected by fluorescent microscopy,RT-PCR and Western blot analysis respectively.The cell proliferation of SW480 cells was tested by MTT assay,the cell cycle was analyzed by flow cytometry,and the invasion ability of SW480 cells were investigated by Transwell-matrigel invasion chambers.Results After transfected into SW480 cells,both the mRNA and protein levels of TGF-?1 were effectively increased (P
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Objective To explore the influence of RNAi-mediated hypoxia-inducible factor 1? (HIF-1?) silencing on the growth and metastasis of colon cancer in nude mice.Methods A total of 30 6-week-old female nude mice were randomly divided into normal saline group (NS group),negative plasmid group and RNA interference group after injected with SW480 subcutaneously to form a colon carcinoma.shRNA-HIF-1? vector was constructed and injected (50 ?g/time,once per 2 d,for 7 times) into the tumor mass of nude mice of RNA interference group to induce RNAi.Normal saline or blank vector was injected into the corresponding nude mice.The changes of HIF-1? were detected with RT-PCR and Western blot analysis.The size of tumors,metastasis rate and the positive rate of lymphnode were compared among different groups.Results In 15 d after RNAi vector injection,the tumor volume of RNAi group [(273.9?14.7) mm3] was much smaller than those of NS group [(1 845.5?91.2) mm3] and negative plasmid group [(1 842.7?115.7) mm3] (P
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AIM: To observe the expression of HIF-1? mRNA, HIF-1?, VEGF and iNOS proteins and to investigate their relationship in h ypoxia-treated SW480 cells. METHODS: HIF-1?, VEGF and iNOS proteins were measured by immuno cytochemistry. Western-blot was used to detect HIF-1? protein. HIF-1? mRNA wa s measured by in situ hybridization. RESULTS: Under hypoxic condition, SW480 cells expressed proteins of HIF-1?, VEGF and iNOS more strongly than that under normoxia condition. How e ver, under hypoxia condition, these three proteins expressed weakly or negativel y when the cells treated with genistein, the inhibitor of HIF-1?. Expressions o f HIF-1? and VEGF proteins in cultured SW480 cells under hypoxic condition were completely or partially inhibited by the addition of SNP but the expression of iNOS was unaffected. Another NO donor NOC5, however, induced the expression of t hese three proteins. L-NAME, a non-specific inhibitor of NOS, inhibited the expr ession of HIF-1?, VEGF and iNOS. The levels of HIF-1? mRNA changed slightly i n different oxygen condition or addition of genistein, NO donor or iNOS inhibitor . CONCLUSIONS: Hypoxia induces the expression of HIF-1?, therefor e upregulates the production of VEGF and iNOS. During hypoxia, SNP inhibits but N OC5 promotes HIF-1? expression, indicating that different NO donor acts on the cells through different mechanisms.