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OBJECTIVE@#To determine whether monotropein has an anticancer effect and explore its potential mechanisms against colorectal cancer (CRC) through network pharmacology and molecular docking combined with experimental verification.@*METHODS@#Network pharmacology and molecular docking were used to predict potential targets of monotropein against CRC. Cell counting kit assay, plate monoclonal assay and microscopic observation were used to investigate the antiproliferative effects of monotropein on CRC cells HCT116, HT29 and LoVo. Flow cytometry and scratch assay were used to analyze apoptosis and cell cycle, as well as cell migration, respectively in HCT116, HT29, and LoVo cells. Western blotting was used to detect the expression of proteins related to apoptosis, cell cycle, and cell migration, and the expression of proteins key to the Akt pathway.@*RESULTS@#The Gene Ontology and Reactome enrichment analyses indicated that the anticancer potential of monotropein against CRC might be involved in multiple cancer-related signaling pathways. Among these pathways, RAC-beta serine/threonine-protein kinase (Akt1, Akt2), cyclin-dependent kinase 6 (CDK6), matrix metalloproteinase-9 (MMP9), epidermal growth factor receptor (EGFR), cell division control protein 42 homolog (CDC42) were shown as the potential anticancer targets of monotropein against CRC. Molecular docking suggested that monotropein may interact with the 6 targets (Akt1, Akt2, CDK6, MMP9, EGFR, CDC42). Subsequently, cell activity of HCT116, HT29 and LoVo cell lines were significantly suppressed by monotropein (P<0.05). Furthermore, our research revealed that monotropein induced cell apoptosis by inhibiting Bcl-2 and increasing Bax, induced G1-S cycle arrest in colorectal cancer by decreasing the expressions of CyclinD1, CDK4 and CDK6, inhibited cell migration by suppressing the expressions of CDC42 and MMP9 (P<0.05), and might play an anticancer role through Akt signaling pathway.@*CONCLUSION@#Monotropein exerts its antitumor effects primarily by arresting the cell cycle, causing cell apoptosis, and inhibiting cell migration. This indicates a high potential for developing novel medication for treating CRC.
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Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proliferación Celular , Metaloproteinasa 9 de la Matriz , Simulación del Acoplamiento Molecular , Ciclo Celular , Receptores ErbB , Apoptosis , Neoplasias Colorrectales/patología , Línea Celular TumoralRESUMEN
Objective @# To investigate the effect of pathogenic bacterium-Porphyromonas gingivalis (P.g) on the proliferation and inflammatory factor expression of human colorectal cancer Caco-2 cells, to determine whether the Janus kinase 2-signal transducers and activators of transcription 3 (JAK2-STAT3) pathway is involved in the regulation of Caco-2 cell proliferation by P.g and to provide an experimental basis for further exploring the relationship between P.g and colorectal cancer. @*Methods @# Caco-2 cells were cultured in vitro, and P.g at different multiplicities of infection (MOIs) (0, 1, 10, 25) was selected to stimulate for 12, 24 and 48 h. The effect of P.g on the proliferation of Caco-2 cells was detected by CCK8. The stimulation time was set as 12, 24 and 48 h. MOI=0 was the control group, and MOI=1, 10 and 25 comprised the experimental group. qRT-PCR and Western blot were used to detect the changes in interleukin-6 (IL-6), interleukin-10(IL-10), JAK2 and STAT3 gene and protein (phosphorylated protein) levels in each group. @*Results @# After P.g infection of Caco-2 cells, P.g had a sustained stimulatory effect on the cells for 12, 24 and 48 h at MOI=1 and MOI=10 compared with the control group. Compared with that in the control group, the expression of pro-inflammatory factor IL-6 and related proliferative pathway protein JAK2 and STAT3 in Caco-2 cells with P.g infection increased in a concentration- and time-dependent manner (P<0.05). Additionally, the expression of IL-10, an anti-inflammatory factor, in Caco-2 cells infected with P.g decreased (P<0.05). After the addition of the JAK2 inhibitor AZ960, the proliferation of Caco-2 cells infected with P.g decreased, and the mRNA expression of STAT3 and JAK2 and the protein expression of p-STAT3 and p-JAK2 decreased (P<0.05). @*Conclusion @#P.g can promote the proliferation of the colorectal cancer cell line Caco-2, and the effect of P.g on Caco-2 cells may promote cell proliferation through the JAK2-STAT3 pathway while promoting the expression of the proinflammatory factor IL-6 and inhibiting the expression of the anti-inflammatory factor IL-10, creating an inflammatory environment conducive to cell proliferation, which may be the mechanism by which P.g affects the proliferation of Caco-2 cells.
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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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Objective To investigate the effect of picropodophyllin (PPP) on proliferation and cell cycle of human colorectal cancer cell line HCT-15 and to clarify the related mechanism. Methods Different concentrations of PPP were used to treat HCT-15 cells, the proliferative activity of HCT-15 cells was detected by cell courting kit-8(CCK-8). Morphological changes of HCT-15 cells were observed under inverted microscope. Flow cytometry was used to detect the changes of HCT-15 cell cycle and Western blotting was used to detect the expression of proliferating cell nuclear antigen (PCNA) and cell cyclinD1. Results The result of CCK-8 assay showed that PPP inhibited the proliferation of HCT-15 cells in a dose-time-dependent manner. Under the inverted microscope, it was found that the HCT-15 cells lost their original shape and became round, the refractive index decreased, and the number of living cells decreased significantly. Flow cytometry showed that the proportion of G
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Objective To investigate the effect of DDX46 gene on the radiosensitivity of colorectal cancer cells and underlying mechanism.Methods SW480 cells transfected with DDX46 RNAi or its empty plasmid by lentivirus were set as experimental group and control group,respectively.After 72 h of transfection,the cells were irradiated with 4 Gy X rays.The cell viabilities of these two groups were detected by CCK-8 assay.The number of γ-H2AX foci and the expressions of some key proteins related to DNA damage repair were detected by immunofluorescence technique and Western blot at 24 h after irradiation.Results At 24 h after 4 Gy irradiation,the cell vitality of experimental group was decreased to (15.02±3.92)％(t=-4.696,P ＜ 0.05) of control and(17.43 ±1.83)％(t=4.844,P＜0.01) of nonirradiated cells,but there was no significant difference between 4 Gy irradiated control cells and nonirradiated cells.Meanwhile,compared with the control group,the ATM protein expression level (t =7.530,P ＜0.01) and the number of γ-H2AX foci (t =-3.108,P ＜0.05) were significantly increased in the experimental SW480 cells,while the expression levels of p-ATM and Rad50 were significantly reduced (t =4.260,4.260,P ＜ 0.05),and the protein levels of DNA-PK in these two groups had tiny difference.Conclusions DDX46 RNA silence increases the radiation sensitivity of SW480 cells by inhibiting ATM activation and DNA repair.
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Objective To explore the lethal action and possible mechanism of RI-1, a RAD51 inhibitor, on MSH2 deficient colorectal cancer cells.Methods The expression of MSH2 protein level was assessed by Western blot, and the sensibility of human colorectal cancer cells to RI-1 (10, 20, 30, 40 and 50 μmol/L)was measured by MTT method.Lentivirus vectors MSH2-shRNA and Neg-shRNA (negative control) were constructed and transfected into HT29 cell.Apoptosis and DNA damage of cells treated with RI-1(40 μmol/L)were detected by flow cytometry and Single cell gel electrophoresis respectively.In addition, the formation of γ-H2AX foci was analyzed by immunofluorescence.Results Compared with control, MSH2-deficient HCT8 cells had obviously apoptosis(P<0.01);in HCT8 and HT29 Shmsh2 cells, tail DNA%, tail length, tail moment and olive tail moment were markedly increased(P<0.05),and the number of γ-H2AX focus were increased(P<0.01).Conclusions RAD51 inhibitor RI-1 selectively kills MSH2 deficient colorectal cancer cells by increasing DNA damage.
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Objective To compare the expression of CD 49f splicing isoforms in different human stem cells and colon cancer cell lines , and construct lentiviral vectors overexpressing specific isoform .Methods The expression of CD49f isoforms in different cells was detected by RT-PCR and real-time PCR.The lentiviral vectors overexpressing CD49f isoforms were constructed by molecular cloning method .The overexpression of CD49f in colon cancer cell line HT29 was confirmed by FCM, Western blot and real-time PCR, the invasive ability was examined by transwell assay.Results CD49f splicing isoform B was highly expressed in H 9 human embryonic stem cell line , while iso-form A expressed in epithelial cells .Both isoform A and B were expressed in mesenchymal cells .CD49f isoforms A and B were also expressed in colorectal cancer cell lines , while HT29 and HCT116 showed higher expression of iso-form A than isoform B , HCT8 and LoVo showed higher expression of isoform B than isoform A .Overexpression of CD49f isoform B greatly increased the invasive ability of HT 29 cells, while isoform A showed no effect .Conclusions The expressions of CD49f splicing isoforms A and B in different types of cells are significantly different , which sug-gests that CD49f isoforms play different biological functions in cells .
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Objective:To investigate whether the overexpression of Oct4B1 gene induces epithelial mesenchymal transition in human colorectal cancer SW480 cells and its possible mechanism.Methods: Experimental group(SW480-Oct4B1):Transfection of SW480 cell lines in colorectal cancer with Oct4B1 overexpression plasmid;Control group(SW480-Oct4B1):negative control plasmid with G418 resistance.Stably transfected cell lines were obtained by G418 culture medium.The two groups were compared with:①Detection of Oct4B1 gene expression in stably transfected cell lines by RT-qPCR;②Scratches and Transwell assays were used to estimate migration and invasion;③Detection of EMT related markers E-cadherin,N-cadherin and Vimentin protein expression by Western blot assay;④Detection of Twist gene and protein expression by RT-PCR and Western blot assays.Results: The transient transfection was confirmed by RT-qPCR and the stable transfected cell lines were obtained from two groups of cells transfected with G418 culture medium.Compared with the control group:①RT-qPCR revealed increased expression of Oct4B1 gene in the experimental group(P<0.01);②Cell migration and invasion were significantly increased(P<0.01);③Epithelial marker:the expression of E-cadherin protein was significantly decreased (P<0.01),interstitial marker:the expression of N-cadherin and Vimentin protein was significantly increased (P<0.01);④Twist mRNA and protein expression were significantly increased(P<0.01).Conclusion: Overexpression of Oct4B1 gene can induce epithelial mesenchymal transition in human colorectal cancer SW480 cells,its molecular mechanism may be related to the promotion of Twist expression.
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AIM:To investigate the role of miR-125b in regulating the sensitivity of CD133+ colorectal cancer cells to cisplatin.METHODS:The expression of miR-125b was detected by RT-qPCR in the routine SW480 cells and CD133+ SW480 cells.Flow cytometry analysis was performed to measure the percentage of CD133+ cell population in the SW480 cell line treated with miR-125b and cisplatin.MTT assay was performed to evaluate the effect of miR-125b on the cisplatin-induced cell death in the CD133+ SW480 cells.Bioinformatics and Western blot were performed to determine whether the expression of HAX-1 was regulated by miR-125b.JC-1 staining, Annexin V staining and Western blot analysis were used to study the pathway of apoptosis in the CD133+ SW480 cells co-treated with miR-125b and cisplatin.RESULTS:The expression of miR-125b was significantly lower in the CD133+ SW480 cells than that in the routine SW480 cells and normal colonic epithelial FHC cells.Treatment with cisplatin alone increased the percentage of CD133+ SW480 cell population.However, miR-125b significantly inhibited the enrichment of CD133+ cell population induced by cisplatin.In addition, the results of MTT assay showed that the anti-tumor effect of cisplatin was significantly enhanced when the miR-125b was transfected into the CD133+ SW480 cells.The results of Western blot indicated that the HAX-1 gene was the target of miR-125b.Furthermore, the apoptosis induced by the combination of miR-125b and cisplatin was dependent on the dysfunction of mitochondrial membrane, leading to the release of cytochrome C into the cytoplasm and the subsequently activation of apoptosis in the CD133+ SW480 cells.CONCLUSION:miR-125b increased the sensitivity of CD133+ colo-rectal cancer cells to cisplatin by down-regulating the expression of HAX-1.
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Objective To investigate the effect of 5-FU on the expression of SHP2 gene in colorectal cancer cells RKO . Methods The human colorectal cancer cell line RKO of logarithmic growth phase was cultured 48 h with 5-FU .The expression of SHP2 was observed by immunofluorescence and Western blotting ;siSHP2 which specifically inhibits the expression of SHP2 was transfected into RKO cells and cultured 48 h with 5-FU .The cell absorbance (A) values were measured with CCK8 and apoptosis was determined by flow cytometry .The sensitivity of RKO to 5-FU and the effect of 5-FU on apoptosis of RKO cells were observed .Results The expression of nuclear protein of SHP2 was improved remarkably after colorectal cancer cell RKO was cultured 48 h with 5-FU .The sensitivity of RKO cells to 5-FU and the cell apoptosis rate induced by 5-FU were decreased after siSHP2 transfection .Conclusion 5-FU exerts anti-cancer activity possibly due to its promoting the apoptosis of RKO cells by influencing the expression of SHP2 .
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Tripolinolate A (TLA) is recently identified as a new compound from a halophyte plant Tripolium vulgare and has been shown to have significant in vitro activity against the proliferation of colorectal cancer and glioma cells. This study was designed to further investigate the effects of TLA on the proliferation of human normal cells, and the apoptosis and cell cycle in colorectal cancer cells, and the growth of tumors in the colorectal cancer-bearing animals. The data obtained from this study demonstrated that: 1) TLA had much less cytotoxicity in the human normal cells than the colorectal cancer cells; 2) TLA remarkably induced apoptosis in the human colorectal cancer cells and blocked cell cycle at G/M phase, and 3) TLA had significant anti-colorectal cancer activity in the tumor-bearing animals.
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Animales , Humanos , Masculino , Ratones , Antineoplásicos Fitogénicos , Química , Apoptosis , Asteraceae , Química , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales , Quimioterapia , Medicamentos Herbarios Chinos , Química , Ésteres , Química , Fase G2 , Ratones Endogámicos BALB C , Fenoles , QuímicaRESUMEN
We identified a novel Akt signaling mechanism that mediates fucoidan-induced suppression of human colon cancer cell (HT29) proliferation and anticancer effects. Fucoidan treatment significantly inhibited growth, induced G1-phase-associated upregulation of p21WAF1 expression, and suppressed cyclin and cyclin-dependent kinase expression in HT29 colon cancer cells. Additionally, fucoidan treatment activated the Akt signaling pathway, which was inhibited by treatment with an Akt inhibitor. The inhibition of Akt activation reversed the fucoidan-induced decrease in cell proliferation, the induction of G1-phase-associated p21WAF1 expression, and the reduction in cell cycle regulatory protein expression. Intraperitoneal injection of fucoidan reduced tumor volume; this enhanced antitumor efficacy was associated with induction of apoptosis and decreased angiogenesis. These data suggest that the activation of Akt signaling is involved in the growth inhibition of colon cancer cells treated with fucoidan. Thus, fucoidan may serve as a potential therapeutic agent for colon cancer.
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Humanos , Apoptosis , Ciclo Celular , Puntos de Control del Ciclo Celular , Proliferación Celular , Neoplasias del Colon , Ciclinas , Inyecciones Intraperitoneales , Fosfotransferasas , Carga Tumoral , Regulación hacia ArribaRESUMEN
Objective To investigate the effect of C225 on DNA repair and molecular pathways in CL187 colorectal cancer cells after irradiated by 125Ⅰ radioactive seeds.Methods In the experiment involved were four groups:control group,100 nmol/L C225 treatment group,125Ⅰ radioactive seeds continuous low-dose rate irradiation group and C225 combined with 125Ⅰ radioactive seeds continuous lowdose rate irradiation group.Cells were collected at 48 h after 4 Gy irradiation,and γH2AX foci/cell and γH2AX foci positive cells were counted with immunofluorescence.At the same time,DNA repair proteins were detected by Western blot.Cells were lyzed immediately after 4 Gy irradiation,and changs in EGFR downstream signaling molecules were detected by Western blot.Results Compared with 125Ⅰ seeds irradiated cells,cells treated with C225 and 125Ⅰ seeds irradiation showed more γH2AX foci per cell (t =8.0,P =0.05),and more γH2AX foci positive cells (t =6.8,P ＜ 0.05) and less expression of Ku70 (t =6.6,P ＜ 0.05) and DNA-PKcs (t =5.6,P ＜ 0.05).Combined with 125Ⅰ-CLDR irradiation,C225 reduced cellular EGFR level(t =4.9,P ＜0.05) and inhibited the activation of Akt(t =5.5,P ＜0.05).Conclusions In the condition of 125Ⅰ seeds irradiation,C225 reduced the expression of Ku70 and DNA-PKcs,inhibited the activation of Akt and attenuated the DNA damage repair capacity in CL187 colorectal cancer cells.
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The electric properties of the plasma membrane is an indicator of cell condition. The simple, and highly effective, normal-phase (NP) and reversed-phase (RP) high-performance liquid chromatography (HPLC) methods assess phospholipid and free unsaturated fatty acid content, respectively. Herein we focus on changes in phospholipid content [phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanoloamine (PE), phosphatidylcholine (PC)] and free unsaturated fatty acid content [arachidonic acid (AA), linoleic acid (LA), a-linolenic acid (ALA), palmitoleic acid (PA)] in the plasma membranes of non-metastatic colorectal cancer cells (pT3 stage, G2 grade). Surface charge density of normal and tumor large intestine tissue was measured by electrophoresis. The surface charge density as a function of pH, acidic (CTA) and basic (CTB-) functional group concentrations and their average association constants with hydrogen (KAH) or hydroxyl (KBOH-) ions were evaluated. Cancer transformation was accompanied by an increase in total phospholipids as well as and increase in CTA, CTB and KBOH whereas the content of free fatty acids and KAH decreased compared with unchanged tumor cells.