Objective:The aim of our study was to investigate the effect of integrin β1 on influencing the sensitivity to chemotherapy of human pulmonary adenocarcinoma A549 cells.Methods:Human pulmonary adenocarcinoma A549 mult...Objective:The aim of our study was to investigate the effect of integrin β1 on influencing the sensitivity to chemotherapy of human pulmonary adenocarcinoma A549 cells.Methods:Human pulmonary adenocarcinoma A549 multicellular spheroids(MCS) were constructed with three dimensional cell culture methods.Cell counting using blood cell counter was employed to detect the sensitivity to ADM of A549 MCS before and after blocking integrin β1;integrin β1 expression of A549 MCS and cell apoptosis was detected by flow cytometry.Results:A549 MCS were successfully established.The integrin β1 expression of A549 MCS elevated with the concentration of ADM(< 0.02 μg/mL).Blocking of integrin β1 lead to higher sensitivity to ADM,and IC50 decreased from 0.19 μg/mL to 0.11 μg/mL,and apoptosis rate increased from(15.81 ± 1.87)% to(30.14 ± 2.89)%.Conclusion:The cell adhesion molecules integrin β1 could influence the sensitivity to chemotherapy of A549 MCS and inhibiting of cell apoptosis might be its mechanism.展开更多
AIM:To investigate the role of profilin-1(PFN1)in gastric cancer and the underlying mechanisms.METHODS:Immunohistochemical analysis,quan-titative real-time polymerase chain reaction(q RTPCR)and Western blot were perfo...AIM:To investigate the role of profilin-1(PFN1)in gastric cancer and the underlying mechanisms.METHODS:Immunohistochemical analysis,quan-titative real-time polymerase chain reaction(q RTPCR)and Western blot were performed to detect PFN1expression in clinical gastric carcinoma and adjacent tissues,and the association of PFN1 expression with patient clinicopathological characteristics was analyzed.PFN1 was knocked down to investigate the role of this protein in cell proliferation and metastasis in the SGC-7901 cell line.To explore the underlying mechanisms,the expression of integrinβ1 and the activity of focal adhesion kinase(FAK)and the downstream proteins extracellular-regulated kinase(ERK)1/2,P38 mitogen-activated protein kinase(MAPK),phosphatidylinositol 3-kinase(PI3K),AKT and mammalian target of rapamycin(m TOR)were measured through Western blot or q RT-PCR analysis.Fibronectin(FN),a ligand of integrinβ1,was used to verify the correlation between alterations in the integrinβ1/FAK pathway and changes in tumor cell aggressiveness upon PFN1 perturbation.RESULTS:Immunohistochemical,Western blot and q RT-PCR analyses revealed that PFN1 expression was higher at both the protein and m RNA levels in gastric carcinoma tissues compared with the adjacent tissues.In addition,high PFN1 expression(53/75,70.4%)was correlated with tumor infiltration,lymph node metastasis and TNM stage in gastric cancer,but not with gender,age,location,tumor size,or histological differentiation.In vitro experiments showed that PFN1knockdown inhibited the proliferation of SGC-7901cells through the induction G0/G1 arrest.Silencing PFN1 inhibited cell migration and invasion and downregulated the expression of matrix metalloproteinase(MMP)-2 and MMP9.Moreover,silencing PFN1 reduced the expression of integrinβ1 at the protein level and inhibited the activity of FAK,and the downstream effectors ERK1/2,P38MAPK,PI3K,AKT and m TOR.FN-promoted cell proliferation and metastasis via the integrinβ1/FAK pathway was ameliorated by PFN1silencing.CONCLUSION:These findings suggest that PFN1 plays a critical role in gastric carcinoma progression,and these effects are likely mediated through the integrinβ1/FAK pathway.展开更多
Hypertrophic differentiation is not only the terminal process of endochondral ossification in the growth plate but is also an important pathological change in osteoarthritic cartilage.Collagen type II(COL2A1)was previ...Hypertrophic differentiation is not only the terminal process of endochondral ossification in the growth plate but is also an important pathological change in osteoarthritic cartilage.Collagen type II(COL2A1)was previously considered to be only a structural component of the cartilage matrix,but recently,it has been revealed to be an extracellular signaling molecule that can significantly suppress chondrocyte hypertrophy.However,the mechanisms by which COL2A1 regulates hypertrophic differentiation remain unclear.In our study,a Col2a1 p.Gly1170Ser mutant mouse model was constructed,and Col2a1 loss was demonstrated in homozygotes.Loss of Col2a1 was found to accelerate chondrocyte hypertrophy through the bone morphogenetic protein(BMP)-SMAD1 pathway.Upon interacting with COL2A1,integrinβ1(ITGB1),the major receptor for COL2A1,competed with BMP receptors for binding to SMAD1 and then inhibited SMAD1 activation and nuclear import.COL2A1 could also activate ITGB1-induced ERK1/2 phosphorylation and,through ERK1/2-SMAD1 interaction,it further repressed SMAD1 activation,thus inhibiting BMP-SMAD1-mediated chondrocyte hypertrophy.Moreover,COL2A1 expression was downregulated,while chondrocyte hypertrophic markers and BMP-SMAD1 signaling activity were upregulated in degenerative human articular cartilage.Our study reveals novel mechanisms for the inhibition of chondrocyte hypertrophy by COL2A1 and suggests that the degradation and decrease in COL2A1 might initiate and promote osteoarthritis progression.展开更多
Mechanical microenvironment can strongly affect the metastatic efficiency of circulating tumor cells.However,the effect of suspension state on their extravasation and the mechanisms involved are still unclear.To explo...Mechanical microenvironment can strongly affect the metastatic efficiency of circulating tumor cells.However,the effect of suspension state on their extravasation and the mechanisms involved are still unclear.To explore the influence of suspension state on extravasation(including adhesion,spreading and transendothelial migration)of breast tumor cells and its relevant molecular mechanism,MDA-MB-231 cells were cultured on poly(2-hydroxyethyl methacrylate)coated 6-well plates to minic the suspension state.Suspension state promoted adhesion,spreading and transendothelial migration of MDA-MB-231 cells to EAhy926 endothelial cells(ECs)monolayer under both the static condition and 0.5 dyne/cm^(2) flow shear stress(FSS).The number of cells adhered to ECs monolayer reached 2.15(static condition,3 d)and 1.67(FSS,3 d)times,and the number of migration reached 10.60 times,respectively,as compared to that in adhesion state.Moreover,MDA-MB-231 cells knockdown of integrin β1 provoked poor adhesion and transendothelial migration,as compared with MDA-MB-231 cells.But it made no difference in cell spreading.Our results implied the increasing expression of integrin β1 induced by suspension culture promoted the adhesion and transendothelial migration of MDA-MB-231 cells,but had no significant influence on their spreading.展开更多
Background: In the previous decade, various benefits and biological activities of royal jelly, applied in alternative and modern medicine, and cosmetics, have been reported. However, the effects of royal jelly extract...Background: In the previous decade, various benefits and biological activities of royal jelly, applied in alternative and modern medicine, and cosmetics, have been reported. However, the effects of royal jelly extract (RJ) on keratinocytes have not been fully elucidated. Objective: The primary objectives of this study were to reveal the effects of RJ on keratinocytes and explore the underlying mechanism. Methods: HaCaT cells, an immortal human epidermis-derived keratinocyte cell line, were used in this study. Laminin α3 (LAMA3), integrin β1 (ITGB1), and hypoxia-inducible factor-1α (HIF-1α) mRNA expression levels were determined using real-time PCR. Cell proliferation rate was measured using a bromodeoxyuridine uptake assay. Results: RJ treatment upregulated LAMA3, ITGB1 and HIF-1α mRNA expression, and accelerated HaCaT cell proliferation. Akt and mTOR inhibitors suppressed the RJ-induced HIF-1α expression and cell proliferation. HIF-1α silencing abrogated RJ-induced LAMA3 and ITGB1 mRNA expression and cell proliferation, whereas LAMA3 silencing and antibody-mediated ITGB1 blockade did not affect the effects of RJ. Conclusion: RJ upregulates LAMA3 and ITGB1 mRNA expression levels by HIF-1α expression enhancement. In addition, RJ accelerates keratinocyte proliferation via Akt/mTOR/HIF-1α/NF-κB signaling pathway. These suggest that RJ is beneficial for anti-aging, as a skin care product ingredient.展开更多
AIM:To clarify the mechanisms of integrin overexpression in negatively regulating the cell cycle control of hepatocellular carcinoma cells SMMC-7721.METHODS: The cell cycle pattern was determined by flow cytometry. Th...AIM:To clarify the mechanisms of integrin overexpression in negatively regulating the cell cycle control of hepatocellular carcinoma cells SMMC-7721.METHODS: The cell cycle pattern was determined by flow cytometry. The mRNA and protein expression levels were assayed by RT-PCR and Western blot, respectively. Stable transfection was performed by Lipofectamine 2000 reagent,and cells were screened by G418.RESULTS: Overexpression of α5β1 or β1 integrin induced S-phase delay in SMMC-7721 cells, and this delay was possibly due to the accumulation of cyclin-dependent kinase inhibitors (CKIs) p21cip1 and p27kip1. The decrease of protein kinase B (PKB) phosphorylation was present in this signaling pathway, but focal adhesion kinase (FAK) was not involved.When phosphorylation of PKB was solely blocked by wortmannin, p27kip1 protein level was increased. Moreover,S-phase delay was recurred when attachment of the parental SMMC-7721 cells was inhibited by the preparation of polyHEME, and this cell cycle pattern was similar to that of β1-7721 or α5β1-7721 cells.CONCLUSION: S-phase delay induced by overexpression of integrin β1 subunit is attributed to the decrease of PKB phosphorylation and subsequent increases of p21cip1 and p27kip1 proteins, and may be involved in the unoccupied α5β1because of lack of its ligands.展开更多
AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721) to human umbilical vein endothelial cells (ECV-304), expression of adhesive molecule integrinβ1 in SMMC-...AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721) to human umbilical vein endothelial cells (ECV-304), expression of adhesive molecule integrinβ1 in SMMC-7721 cells and its contribution to this adhesive course. METHODS: Adhesive force of SMMC-7721 cells to endothelial cells was measured using micropipette aspiration technique. Synchronous G1 and S phase SMMC-7721 cells were achieved by thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronous rates of SMMC-7721 cells and expression of integrinβ1 in SMMC-7721 cells were detected by flow cytometer. RESULTS: The percentage of cell cycle phases of general SMMC-7721 cells was 11.01% in G2/M phases, 53.51% in G0/G1 phase, and 35.48% in S phase. The synchronous rates of G1 and S phase SMMC-7721 cells amounted to 74.09% and 98.29%, respectively. The adhesive force of SMMC-7721 cells to endothelial cells changed with the variations of adhesive time and presented behavior characteristics of adhesion and de-adhesion. S phase SMMC-7721 cells had higher adhesive forces than d phase cells [(307.65±92.10)×10-10N vs(195.42±60.72)×10-10N, P<0.01]. The expressive fluorescent intensity of integrinβ1 in G1 phase SMMC-7721 cells was depressed more significantly than the values of S phase and general SMMC-7721cells. The contribution of adhesive integrinβ1 was about 53% in this adhesive course. CONCLUSION: SMMC-7721 cells can be synchronized preferably in d and S phases with thymine-2-deoxyriboside and colchicines. The adhesive molecule integrinβ1 expresses a high level in SMMC-7721 cells and shows differences in various cell cycles, suggesting integrin β1 plays an important role in adhesion to endothelial cells. The change of adhesive forces in different cell cycle SMMC-7721 cells indicates that S phase cells play predominant roles possibly while they interact with endothelial cells.展开更多
文摘Objective:The aim of our study was to investigate the effect of integrin β1 on influencing the sensitivity to chemotherapy of human pulmonary adenocarcinoma A549 cells.Methods:Human pulmonary adenocarcinoma A549 multicellular spheroids(MCS) were constructed with three dimensional cell culture methods.Cell counting using blood cell counter was employed to detect the sensitivity to ADM of A549 MCS before and after blocking integrin β1;integrin β1 expression of A549 MCS and cell apoptosis was detected by flow cytometry.Results:A549 MCS were successfully established.The integrin β1 expression of A549 MCS elevated with the concentration of ADM(< 0.02 μg/mL).Blocking of integrin β1 lead to higher sensitivity to ADM,and IC50 decreased from 0.19 μg/mL to 0.11 μg/mL,and apoptosis rate increased from(15.81 ± 1.87)% to(30.14 ± 2.89)%.Conclusion:The cell adhesion molecules integrin β1 could influence the sensitivity to chemotherapy of A549 MCS and inhibiting of cell apoptosis might be its mechanism.
文摘AIM:To investigate the role of profilin-1(PFN1)in gastric cancer and the underlying mechanisms.METHODS:Immunohistochemical analysis,quan-titative real-time polymerase chain reaction(q RTPCR)and Western blot were performed to detect PFN1expression in clinical gastric carcinoma and adjacent tissues,and the association of PFN1 expression with patient clinicopathological characteristics was analyzed.PFN1 was knocked down to investigate the role of this protein in cell proliferation and metastasis in the SGC-7901 cell line.To explore the underlying mechanisms,the expression of integrinβ1 and the activity of focal adhesion kinase(FAK)and the downstream proteins extracellular-regulated kinase(ERK)1/2,P38 mitogen-activated protein kinase(MAPK),phosphatidylinositol 3-kinase(PI3K),AKT and mammalian target of rapamycin(m TOR)were measured through Western blot or q RT-PCR analysis.Fibronectin(FN),a ligand of integrinβ1,was used to verify the correlation between alterations in the integrinβ1/FAK pathway and changes in tumor cell aggressiveness upon PFN1 perturbation.RESULTS:Immunohistochemical,Western blot and q RT-PCR analyses revealed that PFN1 expression was higher at both the protein and m RNA levels in gastric carcinoma tissues compared with the adjacent tissues.In addition,high PFN1 expression(53/75,70.4%)was correlated with tumor infiltration,lymph node metastasis and TNM stage in gastric cancer,but not with gender,age,location,tumor size,or histological differentiation.In vitro experiments showed that PFN1knockdown inhibited the proliferation of SGC-7901cells through the induction G0/G1 arrest.Silencing PFN1 inhibited cell migration and invasion and downregulated the expression of matrix metalloproteinase(MMP)-2 and MMP9.Moreover,silencing PFN1 reduced the expression of integrinβ1 at the protein level and inhibited the activity of FAK,and the downstream effectors ERK1/2,P38MAPK,PI3K,AKT and m TOR.FN-promoted cell proliferation and metastasis via the integrinβ1/FAK pathway was ameliorated by PFN1silencing.CONCLUSION:These findings suggest that PFN1 plays a critical role in gastric carcinoma progression,and these effects are likely mediated through the integrinβ1/FAK pathway.
基金supported by the National Natural Science Foundation of China (No.81371907,No.81572134,and No.81802217)the China Postdoctoral Science Foundation (No.2017M622873)+2 种基金the Natural Science Foundation of Guangdong Province,China (No.2018A0303130260,No.2016A030313284,and No.2017A030311008)the Guangzhou Science and Technology Plan (No.201804010057)the Fundamental Research Funds for the Central Universities (No.17ykpy06)
文摘Hypertrophic differentiation is not only the terminal process of endochondral ossification in the growth plate but is also an important pathological change in osteoarthritic cartilage.Collagen type II(COL2A1)was previously considered to be only a structural component of the cartilage matrix,but recently,it has been revealed to be an extracellular signaling molecule that can significantly suppress chondrocyte hypertrophy.However,the mechanisms by which COL2A1 regulates hypertrophic differentiation remain unclear.In our study,a Col2a1 p.Gly1170Ser mutant mouse model was constructed,and Col2a1 loss was demonstrated in homozygotes.Loss of Col2a1 was found to accelerate chondrocyte hypertrophy through the bone morphogenetic protein(BMP)-SMAD1 pathway.Upon interacting with COL2A1,integrinβ1(ITGB1),the major receptor for COL2A1,competed with BMP receptors for binding to SMAD1 and then inhibited SMAD1 activation and nuclear import.COL2A1 could also activate ITGB1-induced ERK1/2 phosphorylation and,through ERK1/2-SMAD1 interaction,it further repressed SMAD1 activation,thus inhibiting BMP-SMAD1-mediated chondrocyte hypertrophy.Moreover,COL2A1 expression was downregulated,while chondrocyte hypertrophic markers and BMP-SMAD1 signaling activity were upregulated in degenerative human articular cartilage.Our study reveals novel mechanisms for the inhibition of chondrocyte hypertrophy by COL2A1 and suggests that the degradation and decrease in COL2A1 might initiate and promote osteoarthritis progression.
基金supported in part by grants from the National Natural Science Foundation of China(11672051)Fundamental Research Funds for the Central Universities(2018CDQYSG0015).
文摘Mechanical microenvironment can strongly affect the metastatic efficiency of circulating tumor cells.However,the effect of suspension state on their extravasation and the mechanisms involved are still unclear.To explore the influence of suspension state on extravasation(including adhesion,spreading and transendothelial migration)of breast tumor cells and its relevant molecular mechanism,MDA-MB-231 cells were cultured on poly(2-hydroxyethyl methacrylate)coated 6-well plates to minic the suspension state.Suspension state promoted adhesion,spreading and transendothelial migration of MDA-MB-231 cells to EAhy926 endothelial cells(ECs)monolayer under both the static condition and 0.5 dyne/cm^(2) flow shear stress(FSS).The number of cells adhered to ECs monolayer reached 2.15(static condition,3 d)and 1.67(FSS,3 d)times,and the number of migration reached 10.60 times,respectively,as compared to that in adhesion state.Moreover,MDA-MB-231 cells knockdown of integrin β1 provoked poor adhesion and transendothelial migration,as compared with MDA-MB-231 cells.But it made no difference in cell spreading.Our results implied the increasing expression of integrin β1 induced by suspension culture promoted the adhesion and transendothelial migration of MDA-MB-231 cells,but had no significant influence on their spreading.
文摘Background: In the previous decade, various benefits and biological activities of royal jelly, applied in alternative and modern medicine, and cosmetics, have been reported. However, the effects of royal jelly extract (RJ) on keratinocytes have not been fully elucidated. Objective: The primary objectives of this study were to reveal the effects of RJ on keratinocytes and explore the underlying mechanism. Methods: HaCaT cells, an immortal human epidermis-derived keratinocyte cell line, were used in this study. Laminin α3 (LAMA3), integrin β1 (ITGB1), and hypoxia-inducible factor-1α (HIF-1α) mRNA expression levels were determined using real-time PCR. Cell proliferation rate was measured using a bromodeoxyuridine uptake assay. Results: RJ treatment upregulated LAMA3, ITGB1 and HIF-1α mRNA expression, and accelerated HaCaT cell proliferation. Akt and mTOR inhibitors suppressed the RJ-induced HIF-1α expression and cell proliferation. HIF-1α silencing abrogated RJ-induced LAMA3 and ITGB1 mRNA expression and cell proliferation, whereas LAMA3 silencing and antibody-mediated ITGB1 blockade did not affect the effects of RJ. Conclusion: RJ upregulates LAMA3 and ITGB1 mRNA expression levels by HIF-1α expression enhancement. In addition, RJ accelerates keratinocyte proliferation via Akt/mTOR/HIF-1α/NF-κB signaling pathway. These suggest that RJ is beneficial for anti-aging, as a skin care product ingredient.
基金Grants from the National Natural Science Foundation of China,No.30000083Shanghai Municipal Government Science and Technology Committee,No.00JC14042
文摘AIM:To clarify the mechanisms of integrin overexpression in negatively regulating the cell cycle control of hepatocellular carcinoma cells SMMC-7721.METHODS: The cell cycle pattern was determined by flow cytometry. The mRNA and protein expression levels were assayed by RT-PCR and Western blot, respectively. Stable transfection was performed by Lipofectamine 2000 reagent,and cells were screened by G418.RESULTS: Overexpression of α5β1 or β1 integrin induced S-phase delay in SMMC-7721 cells, and this delay was possibly due to the accumulation of cyclin-dependent kinase inhibitors (CKIs) p21cip1 and p27kip1. The decrease of protein kinase B (PKB) phosphorylation was present in this signaling pathway, but focal adhesion kinase (FAK) was not involved.When phosphorylation of PKB was solely blocked by wortmannin, p27kip1 protein level was increased. Moreover,S-phase delay was recurred when attachment of the parental SMMC-7721 cells was inhibited by the preparation of polyHEME, and this cell cycle pattern was similar to that of β1-7721 or α5β1-7721 cells.CONCLUSION: S-phase delay induced by overexpression of integrin β1 subunit is attributed to the decrease of PKB phosphorylation and subsequent increases of p21cip1 and p27kip1 proteins, and may be involved in the unoccupied α5β1because of lack of its ligands.
基金Supported by the National Natural Science Foundation of China, No.19972077 and No.10372121
文摘AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721) to human umbilical vein endothelial cells (ECV-304), expression of adhesive molecule integrinβ1 in SMMC-7721 cells and its contribution to this adhesive course. METHODS: Adhesive force of SMMC-7721 cells to endothelial cells was measured using micropipette aspiration technique. Synchronous G1 and S phase SMMC-7721 cells were achieved by thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronous rates of SMMC-7721 cells and expression of integrinβ1 in SMMC-7721 cells were detected by flow cytometer. RESULTS: The percentage of cell cycle phases of general SMMC-7721 cells was 11.01% in G2/M phases, 53.51% in G0/G1 phase, and 35.48% in S phase. The synchronous rates of G1 and S phase SMMC-7721 cells amounted to 74.09% and 98.29%, respectively. The adhesive force of SMMC-7721 cells to endothelial cells changed with the variations of adhesive time and presented behavior characteristics of adhesion and de-adhesion. S phase SMMC-7721 cells had higher adhesive forces than d phase cells [(307.65±92.10)×10-10N vs(195.42±60.72)×10-10N, P<0.01]. The expressive fluorescent intensity of integrinβ1 in G1 phase SMMC-7721 cells was depressed more significantly than the values of S phase and general SMMC-7721cells. The contribution of adhesive integrinβ1 was about 53% in this adhesive course. CONCLUSION: SMMC-7721 cells can be synchronized preferably in d and S phases with thymine-2-deoxyriboside and colchicines. The adhesive molecule integrinβ1 expresses a high level in SMMC-7721 cells and shows differences in various cell cycles, suggesting integrin β1 plays an important role in adhesion to endothelial cells. The change of adhesive forces in different cell cycle SMMC-7721 cells indicates that S phase cells play predominant roles possibly while they interact with endothelial cells.
