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ObjectiveTo explore the effect of Baitouweng Tang （BTWT） on the apoptosis of human colorectal cancer HCT116 cells and decipher the underlying mechanism based on the Hedgehog （Hh） signaling pathway. MethodHCT116 cells were treated with BTWT （25， 50， 100， 200， 500， 750， and 1 000 mg·L-1） for 24 h， and then the cell proliferation was detected by methyl thiazolyl tetrazolium （MTT） colorimetry. Five groups were designed for the treatment of HCT116 cells， including a blank control group， BTWT groups （125， 250， and 500 mg·L-1）， and a positive control （5-fluorouracil， 5-FU， 40 mmol·L-1） group. The cell morphology was observed under an inverted microscope. The migration of the cells was detected by scratch test， and the apoptosis by Hoechest 33324/propidium iodide （PI） staining and flow cytometry. Western blot was employed to determine the protein levels of sonic hedgehog （SHh）， GLI family zinc finger protein 1 （Gli1）， smoothened （Smo）， suppressor of fused （SuFu）， cellular-myelocytomatosis viral oncogene （c-Myc）， and the apoptosis-related proteins B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X protein （Bax）. The quantitative real-time reverse transcription PCR （Real-time PCR） was employed to determine the mRNA levels of Bax， Bcl-2， SHh， Gli1， Smo， SuFu， and c-Myc. ResultCompared with the blank control group， BTWT changed the cell morphology （making the cell become round with dense nucleus）， inhibited the proliferation of HCT116 cells in a dose-dependent manner， decreased the ability of migration （P<0.05， P<0.01）， and increased apoptotic cells. Compared with the blank control group， BTWT （500 mg·L-1） treatment for 24 h up-regulated the protein and mRNA levels of Bax （P<0.05， P<0.01） and down-regulated the protein and mRNA levels of Bcl-2 in HCT116 cells （P<0.05， P<0.01）. Moreover， the treatment down-regulated the mRNA and protein levels of SHh， Gli1， Smo， and c-Myc （P<0.05， P<0.01） and up-regulated the mRNA and protein levels of SuFu （P<0.05， P<0.01）. ConclusionBTWT inhibited the proliferation and migration and induced the apoptosis of colorectal cancer HCT116 cells by down-regulating the Hh signaling pathway.
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Abstract The purpose of this work was to investigate the effects of curcumin on the biological behavior of colorectal cancer cells through the JAK/STAT3 and RAS/MAPK/NF-κB pathways. Human colorectal cancer HCT116 cells were cultured and divided into a control group and low, medium and high-dose curcumin groups (n =5). HCT116 colorectal cancer cells became long-growing cells after incubation and culture at 37°C. The control group was treated with 15μL phosphate-buffered saline, and the low-dose, medium-dose and high-dose curcumin groups were treated with 20, 40 and 80μmol/L curcumin, respectively. All groups were treated with relevant drug intervention, digested and centrifuged for 48h, washed twice with a PBS solution, centrifuged at 1000 rpm for 3 min, and the cells precipitated. The proliferation, apoptosis and growth cycle of cells in each group were observed, and the expressions of the JAK/STAT3 and RAS/MAPK/NF-κB pathways and related proteins in each group were studied. Compared with the curcumin low-dose and medium-dose groups, the proliferation ability of the curcumin high-dose group was significantly decreased (P<0.05). When the low-dose and medium-dose curcumin groups were compared with the high-dose curcumin group, the apoptosis ability was significantly increased (P<0.05). When the low-dose and medium-dose curcumin groups were compared, the growth ratio of the G0/G1 phase in the high-dose curcumin group was significantly increased, and the percentage of the S phase was significantly decreased (P<0.05). Compared with the curcumin low-dose and medium-dose groups, the expression of JAK-STAT3 and RAS/MAPK/NF-κB pathway in the curcumin high-dose group was significantly decreased (P<0.05). The protein expressions of STAT3, RAS, P-P38 and P65 in the curcumin high-dose group were significantly lower than those in the curcumin low-dose and medium-dose groups (P<0.05). Curcumin can inhibit the expression of JAK/STAT3 and RAS/MAPK/NF-κB pathways, block the growth cycle, and inhibit the proliferation and induce apoptosis of colorectal cancer cells, providing a new idea for the clinical treatment of colorectal cancer.
Resumen El objetivo del presente trabajo fue investigar los efectos de la curcumina en el comportamiento biológico de las células del cáncer colorrectal mediante el estudio de las vías JAK/STAT3 y RAS/MAPK/NF-KB. Las células del cáncer colorrectal humano HCT116 se cultivaron y dividieron en un grupo control y en grupos con dosis baja, media y alta (n = 5) de curcumina. Las células de cáncer colorrectal HCT116 se convirtieron en células de crecimiento prolongado después de la incubación y cultivo a 37°C. El grupo de control se trató con 15 μL de solución tampón fosfato salina (PBS) y los grupos de curcumina de dosis baja, media y alta se trataron con 20, 40 y 80 μmol/L de curcumina, respectivamente. Todos los grupos fueron tratados con la intervención farmacológica pertinente, digeridos y centrifugados durante 48 horas, lavados dos veces con solución de PBS, centrifugados a 1000 rpm durante 3 minutos, y las células precipitadas. Se observaron la proliferación, la apoptosis y el ciclo de crecimiento de las células de cada grupo, y fueron estudiados las expresiones de las vías JAK/STAT3, RAS/MAPK/NF-KB y proteínas relacionadas en cada grupo. Comparado con los grupos de la dosis baja y media de la curcumina, disminuyó obviamente la capacidad de proliferación del grupo de la dosis alta de la curcumina (P<0,05). Comparado con los grupos de la dosis baja y media de la curcumina, aumentó de modo significativo la capacidad de la apoptosis del grupo de la dosis alta de la curcumina (P<0,05). Comparado con los grupos de la curcumina de dosis baja y media, aumentó obviamente la proporción del crecimiento de la fase G0/G1 en el grupo de la curcumina de dosis alta y el porcentaje de la fase S disminuyó considerablemente (P<0,05). Las expresiones proteicas STAT3, RAS, P-P38 y P65 en el grupo de la dosis alta de la curcumina fueron evidentemente más bajas que las de los grupos de la dosis baja y media de la curcumina (P<0.05). La curcumina puede inhibir la expresión de las vías JAK/STAT3 y RAS/MAPK/NF-KB, bloquear el ciclo del crecimiento y luego inhibir la proliferación e inducir apoptosis de las células del cáncer colorrectal, lo que brinda una nueva idea para el tratamiento clínico del cáncer colorrectal.
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GB7 acetate is a galbulimima alkaloid obtained from Galbulimima belgraveana.However,information regarding its structure,biological activities,and related mechanisms is not entirely available.A series of spectroscopic analyses,structural degradation,interconversion,and crystallography were performed to identify the structure of GB7 acetate.The MTT assay was applied to measure cell proliferation on human colorectal cancer HCT 116 cells.The expressions of the related proteins were measured by Western blotting.Transmission electron microscopy(TEM),acridine orange(AO)and monodansylcadaverine(MDC)staining were used to detect the presence of autophagic vesicles and autolysosomes.A transwell assay was performed to demonstrate metastatic capabilities.Oxygen consumption rate(OCR)and extracellular acidification rate(ECAR)assays were performed to determine the mitochondrial oxidative phosphorylation(OXPHOS)and glycolysis activity of HCT 116 cells.The data showed that GB7 acetate suppressed the proliferation and colony-forming ability of HCT 116 cells.Pretreatment with GB7 acetate significantly induced the formation of autophagic vesicles and autolysosomes.GB7 acetate upregulated the expressions of LC3 and Thr172 phosphorylated adenosine 5'-monophosphate(AMP)-activated pro-tein kinase α(p-AMPKα),which are key elements of autophagy.In addition,GB7 acetate suppressed the metastatic capabilities of HCT 116 cells.Additionally,the production of matrix metallo-proteinase-2(MMP-2)and MMP-9 was reduced,whereas the expression of E-cadherin(E-cad)was upregulated.Furthermore,GB7 acetate significantly reduced mitochondrial OXPHOS and glycolysis.In conclusion,the structure of the novel Galbulimima alkaloid GB7 acetate was identified.GB7 acetate was shown to have anti-proliferative,pro-autophagic,anti-metastatic,and anti-metabolite capabilities in HCT 116 cells.This study might provide new insights into cancer treatment efficacy and cancer chemoprevention.
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Objective:To observe the effect of Jianpi Xiaoai prescription on long non-coding RNA Hox transcript antisense intergenic RNA （lncRNA HOTAIR）/Janus kinase 2 （JAK2） /signal transducer and activator of transcription 3 （STAT3） signaling pathway and to explore the potential mechanism of Jianpi Xiaoai prescription in suppressing the metastasis of colon cancer. Method:The expression of lncRNA HOTAIR in different cells was analyzed. Following the treatment of HCT116 cells with 10%，15%，and 20% Jianpi Xiaoai prescription -containing serum， the invasive ability of Jianpi Xiaoai prescription on HCT116 cells was assessed by transwell assay. The mRNA expression levels of lncRNA HOTAIR，JAK2，and STAT3 were measured by Real-time polymerase chain reaction （Real-time PCR）， and the protein expression levels of JAK2， phosphorylated STAT3 （p-STAT3） and STAT3 by Western blot. Result:The highest expression of lncRNA HOTAIR was detected in HCT116 cells. Compared with the blank group， each Jianpi Xiaoai prescription group exhibited a decreased number of invasive cells （<italic>P</italic><0.05， <italic>P</italic><0.01）. The relative JAK2 mRNA expression in the middle-dose Jianpi Xiaoai prescription group was down-regulated （<italic>P</italic><0.05）， and the relative lncRNA HOTAIR mRNA expression in the middle- and high-dose Jianpi Xiaoai prescription groups and the relative JAK2 mRNA expression in the high-dose Jianpi Xiaoai prescription group were remarkably down-regulated （<italic>P</italic><0.01）. Compared with the blank group，the relative p-STAT3 protein expression was down-regulated in the middle-dose Jianpi Xiaoai prescription group （<italic>P</italic><0.05）， and the relative JAK2 protein expression in the middle- and high-dose Jianpi Xiaoai prescription groups and the relative p-STAT3 protein expression in the high-dose Jianpi Xiaoai prescription group were remarkably down-regulated （<italic>P</italic><0.01）. Conclusion:Jianpi Xiaoai prescription effectively inhibits the metastasis of colon cancer cells， which may be related to the inhibition of lncRNA HOTAIR/JAK2/STAT3 signaling pathway.
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OBJECTIVE：To study the effects of shikonin on autophagy and apoptosis of human colon cancer HCT 116 cells. METHODS：After treating HCT 116 cells for 48 h with shikonin at 0（blank control ）10，20，40 μg/mL，MTT method was used to detect inhibitory rate of cell proliferation. Flow cytometry was used to detect cell apoptosis rate. RT-qPCR assay and Western blotting were respectively used to detect mRNA and protein expressions of microtubule associated protein light chain 3（LC3）and autophagy-related protein Beclin- 1 and p 62. RESULTS ：Compared with blank control ，after treated with 10，20，40 μ g/mL shikonin for 48 h，proliferation inhibitory rate and apoptosis rate of HCT 116 cells were increased significantly （P＜0.05 or P＜ 0.01）. After treated with 10，20，40 μg/mL shikonin for 48 h，mRNA and protein expressions of LC 3，Beclin-1 and p 62 in HCT116 cells were increased to different extents ；except that mRNA expression of LC 3 was not increased significantly after treated with 10 μg/mL shikonin，the difference were statistically significant in other indexes ，compared with blank control （P＜0.05 or P＜0.01）. CONCLUSIONS ：Shikonin can induce the apoptosis of human colon cancer HCT 116 cells and activate its autophagy pathway.
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OBJECTIVE To investigate the effect of scutellarin on the apoptosis of human colorectal cancer cells via the Hippo signaling pathway in vitro. METHODS MTT colorimetric method was used to detect the influence of scutellar?in on the survival rate of HCT116 cells. And the effect of scutellarin at various concentrations on cell morphology was observed by microscopy. Cell scratch experiment was used to detect the influence of scutellarin on the migration of HCT116 cells. Hoechst33342/PI double staining method was used to detect the effect of scutellarin on the apoptosis of HCT116 cells. Western blotting method was used to assess the action of scutellarin on the expressions of Hippo signal?ing pathway-related proteins Mst1, Lats1, YAP1, p-YAP(Ser127), TAZ, and its downstream effector proteins c-Myc and cyclin D1, as well as apoptosis-related proteins Bcl-2 and Bax in HCT116 cells. RESULTS Scutellarin significantly affected the morphology of HCT116 cells and reduced the survival rate of HCT116 cells. Hoechst33342/PI double stain?ing showed that scutellarin effectively induced the apoptosis of HCT116 cells. Western blotting analysis showed that the expression levels of Hippo signaling pathway-related proteins Mst1, Lats1, YAP1, TAZ and its downstream effector pro?teins c-Myc, cyclin D1 were down-regulated in a concentration-dependent manner by scutellarin, and the expression of p-YAP (ser127) was up-regulated. Moreover, scutellarin substantially lessened the expression level of apoptosis-related protein Bcl-2, and promoted the protein level of Bax. CONCLUSION Scutellarin may inhibit the proliferation and migra?tion of HCT116 cells, while induce its apoptosis, potentially by activation of Hippo signaling pathway.
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Objective:To observe the effect of polyphyllin Ⅰ on the expressions of forkhead box Q1（FOXQ1）and epithelial-mesenchymal transition (EMT)-related factors, in order to explore the possible mechanism of polyphyllin Ⅰ in inhibiting the metastasis of colon cancer. Method:After the treatment with 1.25，2.50 μmol·L-1 polyphyllin Ⅰ on HCT116 cells, Western blot and Real-time PCR were used to detect the expressions of FOXQ1，E-cadherin，Vimentin protein and mRNA. Result:Compared with the blank group, relative expressions of FOXQ1 protein and mRNA in low-concentration polyphyllin Ⅰ group were decreased, while relative expression of E-cadherin mRNA was increased, the differences were not statistically significant, and relative expressions of Vimentin protein and mRNA in low-concentration polyphyllin Ⅰ group were decreased (P<0.05)， and relative expression of E-cadherin protein in low-concentration polyphyllin Ⅰ group was increased (P<0.01). Compared with the blank group, relative expressions of FOXQ1, Vimentin protein and mRNA in high-concentration polyphyllin Ⅰ group were decreased，while relative expressions of E-cadherin protein and mRNA were increased (P<0.05, P<0.01). Compared with low-concentration polyphyllin Ⅰ group, relative expressions of Vimentin protein and mRNA in high-concentration polyphyllin Ⅰ group were decreased, but the difference was not statistically significant, relative expressions of E-cadherin protein and mRNA in high-concentration polyphyllin Ⅰ group were increased, whereas relative expressions of FOXQ1 protein and mRNA were decreased (P<0.05, P<0.01). Conclusion:Mechanism of polyphyllin Ⅰ inhibiting the metastasis of colon cancer may be related to the decrease of FOXQ1 and Vimentin expressions, and the up-regulation of E-cadherin.
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Objective:To investigate the mechanism of Jianpi Yangzheng recipe in inhibiting aerobic glycolysis by down-regulating the expression of pyruvate kinase isoenzyme M2 (PKM2) protein, in order to promote apoptosis and inhibite epithelial-mesenchymal transition（EMT）in HCT116 cells of colorectal cancer. Method:The effect of different concentrations of Jianpi Yangzheng recipe on HCT116 cell proliferation was detected by methylthiazolyldiphenyl-tetrazolium bromide（MTT）colorimetry. Flow cytometry was used to detect the effect of different concentrations of Jianpi Yangzheng recipe（2.0, 4.0, 8.0 g·L-1） on HCT116 cell apoptosis. The effect of Jianpi Yangzheng recipe（2.0, 4.0, 8.0 g·L-1） on the migration and invasion ability of HCT116 cells was observed by cell scratch and cell invasion assay (Transwell). The effect of different concentrations of Jianpi Yangzheng recipe（2.0, 4.0, 8.0 g·L-1） on glycolysis metabolism of HCT116 cells were detected by lactic acid (LD) test kit and glucose assay kit, respectively. Western blot was used to detect the expressions of apoptosis-related proteins, like B lymphocyte tumor-2 gene (Bcl-2), Bcl-2 related X protein (Bax) and EMT-related proteins, like epithelial cadherin (E-cadherin)，neurogenic cadherin（N-cadherin）, Vimentin, and PKM2, the key protein of glycolysis, in each group. Result:MTT assay showed that, compared with the blank group, HCT116 cells were treated with Jianpi Yangzheng recipe for 48 h. With the increase of drug concentration, the inhibitory effect of Jianpi Yangzheng recipe on the proliferation of HCT116 cells was also increased; and when the concentration was 4.0 g·L-1, the inhibition rate of HCT116 cells was about 53.87%. Therefore, 2.0，4.0，8.0 g·L-1 were selected as low, medium and high-dose groups for the study. The cell flow cytometry results indicated that, compared with the blank group, the low, medium and high-dose groups all significantly induced the apoptosis of HCT116 cells (P<0.05), and the effect in inducing apoptosis was more obvious with the increase of drug concentration (P<0.05). Cell scratch and Transwell showed that, compared with the blank group, all the groups had an inhibitory effect on migration and invasion of HCT116 cells (P<0.05), and the effect was more significant with the increase of drug concentration (P<0.05). The determination of lactic acid and glucose indicated that compared with the blank group, with the increase of drug concentration, the amount of lactic acid produced by cells in each group gradually decreased (P<0.05), while the glucose dosage also gradually decreased (P<0.05). Western blot showed that, compared with the blank group, the protein expressions of E-cadherin and Bax were up-regulated in groups with different concentrations, whereas the protein expressions of N-cadherin, Vimentin, Bcl-2 and PKM2 were down-regulated (P<0.05). Conclusion:Jianpi Yangzheng recipe can effectively induce the apoptosis of HCT116 cells and inhibit EMT in colorectal cancer. The possible mechanism may be related to the inhibition of aerobic glycolysis pathway of HCT116 cells by down-regulating PKM2 protein expression.
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Objective To explore raddeanin A(RA) inhibiting the proliferation of HCT116 cells and its related mechanism.Methods The MTT method was used to observe the RA inhibition on the proliferation of gastric cancer cells HCT116.Flow cytometry was used to detect the RA effects on cell apoptosis.Transmission electron microscope(TEM) was used to observe RA induced autophagy.Western blot was used to test expression of apoptosis and autophagy related proteins.Results RA could significantly inhibit the proliferation of HCT16 cells with concentration and time dependence;double layer membrane of autophagosome was detected by TEM;FCM showed that RA induced apoptosis in HCT116 cells,moreover the apoptosis rate was increased with the concentration increase;Western blot showed that the expression of autophagy related proteins(Beclin1 and LC3) was increased,the expression of apoptosis inhibition protein Bcl-2 was decreased.On the contrary,the expression of apoptosis promotion proteins(Bax,Cleaved-caspase-3,Cleaved-PARP) was increased,the expression of mTOR protein in the mTOR signal pathway was increased,while the p-mTOR protein expression was decreased.When the mTOR pathway inhibitor rapamycin(RAPA) was added to cells,Beclin-1,LC3,Cleaved-caspase-3,Cleaved-PARP proteins were increased,and apoptosis rate was also increased.Conclusion RA can inhibit the proliferation of HCT116 cells and can induce cellular autophagy and apoptosis,its mechanism may be realized by regulating the mTOR signal pathway.
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Aim To investigate the anti-proliferation effect of honokial on human colon cancer cells HCT116 and the possible mechanism. Methods Crystal stai-ning and flow cytometry assay were introduced to test the proliferation inhibitory and cell cycle arrested effect of honokial on HCT116 cells. Western blot was used to analyse the expression of PCNA and induction effect of apoptosis. Western blot and PCR assay were conducted to assess the change of BMP7. Via exogenous adenovi-ruses of BMP7 and its antibody combined with honoki-al,crystal violet staining and Western blot were used to analyse the effect on HCT116 cells. Results Honoki-al inhibited the growth of HCT116 in a time-and con- centration-dependent way,induced apoptosis and arres-ted at G2 phase; Western blot assay showed honokial up-regulated the level of PCNA and BMP7,and down-regulated the expression of Bcl-2; Western blot result also showed that exogenous adenoviruses of BMP7 en-hanced the effect of honokial on proliferation inhibition and apoptosis, while BMP7 antibody reversed such effects of honokial. Conclusion Honokial can inhibit the proliferation and induce apoptosis on HCT116 cells,which may be mediated by the up-regulation of BMP7.
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Objective To investigate the effects of ZNF331 overexpression on proliferation and apoptosis of human colon cancer cell HCT116, and the relevant apoptotic mechanism.Methods The lentivirus vector of overexpressed ZNF331,Flag-pLV-Neo-ZNF331,was constructed and packaged.HCT116/p53 +/+(wild type p53)and HCT116/p53 -/-(deficient p53)cells were infected.Clones with ZNF331 overexpression were identified by Western blotting.Cell proliferation assay,colony formation assay and flow cytometry analysis were used to examine the effects of ZNF 331 on cell proliferation and apoptosis.Immunoprecipitation,luciferase reporter gene assay and real-time PCR were performed to detect interactions between ZNF331 and p53, p53 transcriptional activity and the expression of p 53 apoptotic target genes, respectively.Results The lentivirus vector of overexpressed ZNF 331 was successfully generated.Stable clones of ZNF331 overexpression were established.ZNF331 showed no significant effect on cell proliferation of HCT 116/p53 +/+, but inhibited cell proliferation of HCT116/p53 -/-(P<0.01).ZNF331 could interact with p53,dose-dependently inhibit the transcriptional activity of p53 and downregulate the mRNA levels of pro-apoptotic p53 target genes, Puma and p53AIP1 (P<0.05).ZNF331 could suppress p53-induced apoptosis(P <0.01).Conclusion The influence of ZNF331 overexpression on colon cancer cell proliferation is dependent on p 53 status.ZNF331 overexpression can suppress colon cancer cell apoptosis by interacting with p 53 and inhibiting the transcriptional activity of p 53.
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Aim To study the relationship between the anti-proliferation effect of tetrandrine(Tet) and TGF-β1 in human colon cancer cells.Methods Cell viability assay, Western blot, flow cytometry and Annexin Ⅴ-EGFP staining were introduced to analyze the anti-cancer effect of Tet on HCT116 cells.Real-time PCR, Western blot,cell viability and immunofluorescent were employed to determine the relationship between the anti-cancer effect of Tet and TGF-β1 in HCT116 cells, the relationship between the anti-cancer effect of Tet and PI3K/Akt on HCT116 cells, and how TGF-β1 mediated the anti-cancer effect of Tet on HCT116 cells.Results Compared with the control groups, Tet apparently inhibited the proliferation, and induced cell cycle arrest at G1 phase and apoptosis in HCT116 cells.Tet greatly up-regulated the expression of TGF-β1 either the mRNA or protein level, and exogenous expression of TGF-β1 potentiated the anti-cancer effect of Tet in HCT116 cells, while TGF-β1 inhibitor attenuated it notably.Tet decreased the phosphorylation of Akt1/2/3, but no apparent effect was observed on total protein level of Akt1/2;PI3K inhibitor enhanced the anti-cancer effect of Tet in HCT116 substantially.Exogenous expression of TGF-β1 enhanced the Tet-induced decrease phorphorylation of Akt1/2/3, which was partly reversed by TGF-β1 inhibitor in HCT116 cells.Meanwhile, knockdown of PTEN elevated the level of phorphorylated Akt1/2/3, which was abolished by the exogenous expression of TGF-β1 in HCT116 cells.Conclusion Tet may be a potent candidate drug for colon cancer treatment, and the anti-cancer effect of Tet may be partly mediated by up-regulating TGF-β1 to inactivate PI3K/Akt signal.
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Objective To investigate the effect of evodiamine on proliferation of HCT-116 in balb/c nude mice, and to explore possible mechanism. Methods HCT-116 cells were injected into the right armpit of 4 week old Balb/c nude mice, the feeding had been executed at the time of the diameter of the xenografted tumor reached 0.5 cm,at the dose of 3 mg/kg (Evo), body weight and tumor diameter had been tested every three days, and made the curve of body weight and tumor diameter of the mice. All the mice were sacrificed after 22 days of feed-ing,and harvested the xenografted tumors. The morphological difference of the two groups tumor were identified by HE staining,the expression of HDAC3, NF-κB and p53 protein were detected by IHC and Western blot. Results Compared with the control groups, the volume and weight of the tumors in Evo groups were significantly lighter, and the body weight of the nude mice were heavier,the tumor cells in Evo groups were shrink,deeply staining nu-celus and their abnormal mitoses were fewer,the expression of NF-κB and p53 were increased but HDAC3 was de-creased in xenografted tumors treated with Evo(P<0.05). Conclusions Evo can change the expression of NF-κB and p53 by down-regulating HDAC3,and inhibit the proliferation of HCT-116 cells line in vivo.
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Objective To investigate the effect of evodiamine on proliferation of HCT-116 in balb/c nude mice, and to explore possible mechanism. Methods HCT-116 cells were injected into the right armpit of 4 week old Balb/c nude mice, the feeding had been executed at the time of the diameter of the xenografted tumor reached 0.5 cm,at the dose of 3 mg/kg (Evo), body weight and tumor diameter had been tested every three days, and made the curve of body weight and tumor diameter of the mice. All the mice were sacrificed after 22 days of feed-ing,and harvested the xenografted tumors. The morphological difference of the two groups tumor were identified by HE staining,the expression of HDAC3, NF-κB and p53 protein were detected by IHC and Western blot. Results Compared with the control groups, the volume and weight of the tumors in Evo groups were significantly lighter, and the body weight of the nude mice were heavier,the tumor cells in Evo groups were shrink,deeply staining nu-celus and their abnormal mitoses were fewer,the expression of NF-κB and p53 were increased but HDAC3 was de-creased in xenografted tumors treated with Evo(P<0.05). Conclusions Evo can change the expression of NF-κB and p53 by down-regulating HDAC3,and inhibit the proliferation of HCT-116 cells line in vivo.
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Objective To establish a stable animal model of transplanted human colorectal cancer.Methods BALB/c nude mice were randomly divided into orthotopic colorectal model group and subcutaneous inoculation model group by random number table,and separately inoculated with 0.1 ml human colon cancer cell HCT116 under the density of 2×107/ml into the orthotopic colorectal (with the self-made inoculator) and right forelimb pit subcutaneously.The mice were observed for 60 days to compare the tumor formation and tumor growth in the two groups.Results The tumor formation rate of all 18 animals was 100 ％ (18/18) in the orthotopic colorectal group.The average tumor weight was (2.78±1.86) g and the average survival time was (45.00±11.99) d.The tumor formation rate was 27.78 ％ (5/18) in the subcutaneous inoculation group.The average tumor weight was (1.74±0.82) g,and the average survival time was (60.00±0.00) d.Conclusion 0.1 ml (2×107/ml) human colon cancer cell suspension HCT116 inoculated into BALB/c nude mice orthotopic colorectal with self-made inoculator could establish human colorectal cancer animal model successfully.
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PURPOSE: To determine CD133+ cells defined as cancer stem cells (CSCs) in colon cancer, we examined whether CD133+ clones in HCT116 demonstrate known features of CSCs like sphere-forming ability, chemodrug-resistance, and metastatic potential. METHODS: Magnetic cell isolation and cell separation demonstrated that <1% of HCT116 cells expressed CD133, with the remaining cells being CD133- clones. In colon cancer cells, radioresistance is also considered a CSC characteristic. We performed clonogenic assay using 0.4 Gy γ-irradiation. RESULTS: Interestingly, there were no differences between HCT116 parental and HCT116 CD133+ clones when the cells comprised 0.5% of the total cells, and CD133- clone demonstrated radiosensitive changes compared with parental and CD133+ clones. Comparing gene expression profiles between sphere-forming and nonforming culture conditions of HCT116 subclones by whole RNA sequencing failed to obtain specific genes expressed in CD133+ clones. CONCLUSION: Despite no differences of gene expression profiles in monolayer attached culture conditions of each clone, sphere-forming conditions of whole HCT116 subclones, parental, CD133+, and CD133- increased 1,761 coding genes and downregulated 1,384 genes related to CSCs self-renewal and survival. Thus, spheroid cultures of HCT116 cells could be useful to expand colorectal CSCs rather than clonal expansion depending on CD133 expressions.
Subject(s)
Humans , Cell Separation , Clinical Coding , Clone Cells , Colonic Neoplasms , HCT116 Cells , Neoplastic Stem Cells , Parents , RNA , Sequence Analysis, RNA , TranscriptomeABSTRACT
Aim To investigate the inhibitory effect of Evodiamine on JAK2/STAT3 signal pathway in human colorectal cancer cell line HCT-116 . Methods Cells were cultured with 6. 0 μmol·L-1 Evodiamine for 2, 4 and 6 h, respectively. Cell nuclear morphology was detected by Hoechst staining and protein expression levels of JAK2 , p-JAK2 , STAT3 and p-STAT3 were examined by Western blot. Cells were treated with dif-ferent concentrations of AG490 for 48 h to select proper working concentration and cells treated with 6 μmol · L-1 EVO and 50 μmol · L-1 AG490 to compare the modulatory effect of EVO with AG490 on JAK2/STAT3 signal pathway. Results Hoechst staining revealed that Evodiamine could induce cells apoptosis, chroma-tin condensation gathered and typical apoptotic mor-phological changes in a time-dependent manner;West-ern Blot suggested that EVO could inhibit p-STAT3 significantly. After treatment with AG490, JAK2/STAT3 signal pathway was inactivated, the inhibitory effect of EVO on p-STAT3 was stronger than that of AG490 , while EVO combined with AG490 could fur-ther inhibit the expression of p-STAT3 significantly. Conclusions The anticancer effect of Evodiamine is mainly mediated by the modulation of JAK2/STAT3 signal pathway in HCT-116 cells.
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EZH2 is over-expressed in human colon cancer and is closely associated with tumor proliferation, metastasis and poor prognosis. Targeting and inhibiting EZH2 may be an effective therapeutic strategy for colon cancer. 3-Deazaneplanocin A (DZNep), as an EZH2 inhibitor, can suppress cancer cell growth. However, the anti-cancer role of DZNep in colon cancer cells has been rarely studied. In this study, we demonstrate that DZNep can inhibit the growth and survival of colon cancer HCT116 cells by inducing cellular senescence and apoptosis. The study provides a novel view of anti-cancer mechanisms of DZNep in human colon cancer cells.
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AIM:To identify and analyze tyrosine-phosphorylated proteins regulated by protein tyrosine phos-phatase-like A domain containing protein 1 ( PTPLAD1) in colon cancer cells by phosphoproteomics.METHODS: The expression of PTPLAD1 in colon cancer cell line HCT-116 was knocked down by small interfering RNAs, and the differenti-al expression of tyrosine-phosphorylated proteins in response to the konckdown of PTPLAD1 in HCT-116 cells was identified by stable isotope labeling with amino acid in cell culture ( SILAC) , coupled with the tyrosine phosphorylation antibody im-munoprecipitation and LC-MS/MS analysis.The Ingenuity Pathway Analysis ( IPA) software was employed for bioinformat-ics analysis on the differentially-expressed proteins.RESULTS:A total of 20 differentially-expressed tyrosine-phosphoryla-ted proteins were identified by MS, including 8 markedly up-regulated and 10 evidently down-regulated proteins.IPA soft-ware suggested that these proteins were mainly associated with the disease of cancer, tissue development and function, and cell death and survival.CONCLUSION:We successfully identified PTPLAD1-regulated differentially-expressed tyrosine-phosphorylated proteins in colon cancer cell line HCT-116.Our analysis suggests that PTPLAD1-regulated proteins in colon cancer are closely correlated with colon cancer.
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Objective To investigate the expression of BRAF-activated long non-coding RNA (BANCR) in colorectal cancer,and the influence of BANCR on the biological function of HCT116 cells.Methods Fifty-six samples of colorectal cancer specimen (including the cancer tissues and precancerous tissues) were obtained at the First Affiliated Hospital of Nanjing Medical University from March 2012 to June 2013.The expressions of BANCR in all the specimens were detected by qRT-PCR (28 cases in the BANCR-high expression group and 28 cases in the BANCR-low expression group).The relationship between the expressions of BANCR and the clinicopathological factors of colorectal cancer was analyzed.The HCT116 cells were divided into 4 groups after interfering BANCR with lentiviral-mediated shRNA-1 and shRNA-2:interference group 1 (HCT116 cells transfected with LV-shRNA-1),interference group 2 (HCT116 cells transfected with LV-shRNA-2),negative control group (HCT116 cells transfected with lentivirus vector with nonsense sequence) and blank control group (HCT116 cells cultured in RPMI 1640 medium).The proliferation,apoptosis and migration of HCT116 cells in the 4 groups were detected by CCK-8 assay,flow cytometry and Transwell assay,respectively.The comparison between the 2 groups was analyzed by u test,and multiple groups were compared by one-way analysis of variance,repeated measurement analysis of variance and LSD-t test.Multivariate analysis was done by Logistic regression model.The difference between categorical data was compared by chi-square test.Results The relative expression of BANCR in the cancer tissues was 1.6 ± 0.4,which was significantly higher than 0.9 ± 0.7 of the precancerous tissues (u =1 020.000,P ＜ 0.05).The result of univariate analysis showed that the high expression of BANCR was correlated with the lymph node metastasis and tumor stage (x2 =4.595,7.487,P ＜ 0.05).The result of multivariate analysis showed that lymph node metastasis and tumor stage (stage Ⅲ-Ⅳ) were the independent risk factors influencing the high expression of BANCR(OR =4.000,5.914,95％ CI:1.230-12.900,1.685-20.760,P ＜ 0.05).The relative expressions of BANCR of the interference group 1,interference group 2,negative control group and the blank control group were 0.25 ±0.04,0.20±0.06,0.96 ±0.04,0.98 ±0.03,with significant difference among the 4 groups (F =271.610,P ＜ 0.05).The cell proliferation rates at day 6 of the interference group 1,interference group 2 and the negative control group were 80.6％ ± 7.6％,81.2％ ± 5.1％ and 87.9％ ± 13.6％,with no significant difference among the 3 groups (F =0.559,P ＞ 0.05).The apoptotic rates of the interference group 1,interference group 2,negative control group and the blank control group were 4.7％ ± 1.7％,5.1％ ± 1.1％,3.1％ ± 0.6％ and 2.8％ ± 0.9％,with no significant difference among the 4 groups (F =2.881,P ＞ 0.05).The numbers of transmembrane cells of the interference group 1,interference group 2,negative control group and the blank control group were 135 ± 29,107 ± 18,240 ± 24 and 245 ± 22,with significant difference among the 4 groups (F =45.194,P ＜ 0.05).Conclusions BANCR was overexpressed in the HCT116 cells,and the BANCR overexpression was correlated with lymph node metastasis and tumor stage.BANCR can promote the migration of HCT116 cells.BANCR could be an important biomarker for the diagnosis and prognosis of colorectal cancer.