ABSTRACT
Objective To explore the effects from the synergy of substrate stiffness and hypoxia on epithelial mesenchymal transition (EMT) of colon cancer cells SW480 by simulating the microenvironment of human colon cancer tissues. Methods Polyvinyl alcohol gels with different stiffness ( 4. 5, 20, 40 kPa) were prepared to simulate the stiffness of each part of colon cancer tissues. The morphological change of cells on substrate with different stiffness was detected under simulated hypoxia ( CoCl2 ) environment. The expression of hypoxia inducible factor (HIF-1α), and EMT markers E-cadherin, Vimentin, Snail 1 were detected by Western blot. The mRNA expression of E-cadherin, Vimentin, Snail 1, matrix metalloproteinase-2 ( MMP-2), and MMP-9 was detected by quantitative real-time PCR ( qRT-PCR). Results Under simulated hypoxia environment, with the increase of substrate stiffness, the SW480 cells spreading area increased, and transformed from round shape into irregular polygon. The EMT of SW480 could be enhanced through up-regulating expression of Vimentin, Snail 1, MMP-2, MMP-9, and down-regulating expression of E-cadherin. Conclusions This study is important for exploring the synergistic effect of substrate stiffness and hypoxia on the EMT of colon cancer cells as well as the molecular mechanism.
ABSTRACT
Substrate stiffness is one of the important mechanical parameters of extracellular matrix. Different substrate stiffness will affect the proliferation, movement and phenotype of tumor cells. It is found that the change of substrate stiffness is also related to the chemotherapy and radiotherapy sensitivity of tumor cells. Substrate stiffness can cause changes in the characteristics of tumor stem cells, which are related to the activation or inactivation of some signaling pathways, mainly involving ABCG2 protein and Akt/mTOR/Sox2 signaling pathway. Further study on the role of substrate stiffness in radiotherapy, chemotherapy and targeted therapy of malignant tumor is expected to provide basis for the clinical treatment of malignant tumor.
ABSTRACT
The elastic stress and viscous shear stress experienced by the vessel wall under pulse blood pressure and blood flow and the mechanical properties of the substrate constitute the in vivo mechanical niches of vascular cells, and these mechanical stimuli are involved in regulating the biological responses of vascular cells and inducing the remodeling and pathological changes of vascular tissues. Although many experimental studies on vascular mechanobiology have been reported, the quantitative correlation between the mechanical stimuli of in vitro experiments and the physiological and pathological conditions of blood vessels remains to be elucidated. This paper summarized the quantitative evaluation method of in vivo mechanical niches of vascular cells from the viewpoint of biomechanics, and then focused on effects of the physiological locations and aging on mechanical behaviors of the vessel wall. This paper also explored the physiological and pathological characteristics of the cellular mechanical niches and their implications for current vascular mechanobiological studies.
ABSTRACT
Objective To investigate the cause of differences in confluent growth between hepatic(L02) and hepatoma carcinoma(HCCLM3) cells by comparing responses of the two cells to different substrate stiffness (0.5, 4 kPa and glass). MethodsThe real-time photomicrography, immunofluorescence staining, flow cytometry, and Western Blotting techniques were respectively employed to observe the morphological characteristics, the cytoskeleton conformation and the distribution of E-cad of confluent L02 and HCCLM3 cells on different substrates, and test the changes in expression of E-cad, Integrinβ1 and p-Src. Results (1) Confluent L02 cells displayed a round or cubic shape, while HCCLM3 cells showed a polygon shape. The morphology of HCCLM3 cells were spread and polarized more obviously than that of L02 cells. With the increase of substrate stiffness, the variation of L02 cells with time was smaller than that of HCCLM3 cells. (2) The cytoskeleton of confluent L02 cells showed a ring-like conformation under the cortex, and E-cad was located at the cell-cell contact sites. However, the ring-like cytoskeleton of HCCLM3 cells was incomplete and distributed radially along the basement, while E-cad was dispersed in cytoplasm. (3) As the substrate stiffness increased, expression of E-cadherin in both L02 and HCCLM3 cells was significantly decreased (P<0.01), while the level of p-Src and integrinβ1 was increased significantly, with greater changes in HCCLM3 cells than in L02 cells. Conclusions The assembling of cortical ring-like cytoskeleton was positively correlated with the location of E-cad at the cell-cell contact sites. The substrate stiffness showed a more obvious impact on the balanced regulation between cadherin and integrin mediated adhesion system of hepatocarcinoma cells than that of hepatic cells.
ABSTRACT
Objective To explore the synergistic effects of substrate stiffness and cytokine TGF-β1 on phenotypic transformation of hepatocytes by establishing an in vitro culture model with the substrate stiffness that is relevant to hepatic cells physiologically and pathologically. Methods Immunofluorescence and Western blotting were adopted to observe the morphological adjustment, motion characteristics, cytoskeleton arrangement of hepatocytes on polyacrylamide substrates with different stiffness, as well as the changes in expression of integrin and phenotypic markers E-cadherin, albumin and alpha-smooth muscle actin (α-SMA). Image analysis software was also used for quantitative study on the obtained data. Results On the 3.6 kPa substrates, the scattered single cells were actively deformed and relocated, but the bulk cell population had little change in polarization and microfilament organization. Muscle actin was assembled as cortical ring in cell periphery. There was more abundant expression of E-cadherin and albumin, but less expression of integrin and α-SMA in TGF-β1 treated group as compared to the control group. On the 30 kPa substrates, the motion and deformation of cells were not so active, and expression of both E-cadherin and albumin in TGF-β1 treated group was decreased, while that of α-SMA was increased as compared to the control group. For 30 kPa and 3.6 kPa control groups and 30 kPa and 3.6 kPa TGF-β1 treated groups, expression of both E-cadherin and albumin was reduced (P<0.05), but that of alpha-SMA was increased (P<0.05), while no significant differences were found in both 10 kPa control group and TGF-β1 treated group, as well as in 30 kPa and 3.6 kPa control groups and TGF-β1 treated groups. Conclusions The increase of substrate stiffness can induce transformation of hepatocyte phenotype and promote the influence of TGF-beta 1 on behavior of hepatocyte metabolism.
ABSTRACT
Objective To investigate the effects of substrate stiffness on the adhesion, spreading and migration of hepatocellular carcinoma cells as well as the regulation of cytoskeleton assembly and integrinβ1 expression, and to explore the role of substrate mechanical properties in the metastasis of hepatocellular carcinoma cells. Methods The polyarcylamide gel with different stiffness was achieved by varying the relative ratio of acrylamide to bis acrylamide. The substrate surface was cross linked with extracellular matrix molecules for cell adhesion. The adhesion, spreading and migration of hepatocellular carcinoma cells on substrates with different stiffness were recorded by phase contrast microscope and made quantitative analysis by Image J software. The cytoskeleton assembly on substrates with different stiffness was detected by immunofluorences assay, and the expression of integrinβ1on different substrates was measured by flow cytometer. Results The rigid substrate enhanced the adhesion and spreading of hepatocellular carcinoma cells in shortened time. Neither the soft (1.1 kPa) nor over rigid (glass) substrate facilitated the migration of hepatocellular carcinoma cells, and the maximum migration velocity was found on the substrate with moderate stiffness（10.7 kPa）. The rigid substrate could promote cytoskeleton assembly and integrinβ1 expression. Conclusions The effects of substrate stiffness on adhesion, spreading and migration of hepatocellular carcinoma cells are regulated by the cytoskeleton assembly and integrin expression.