Gelsolin affects the migratory ability of human colon adenocarcinoma and melanoma cells

AimsFormation of different protrusive structures by migrating cells is driven by actin polymerization at the plasma membrane region. Gelsolin is an actin binding protein controlling the length of actin filaments by its severing and capping activity. The main goal of this study was to determine the effect of gelsolin expression on the migration of human colon adenocarcinoma LS180 and melanoma A375 cells.Main methodsColon adenocarcinoma cell line LS180 was stably transfected with plasmid containing human cytoplasmic gelsolin cDNA tagged to enhanced green fluorescence protein (EGFP). Melanoma A375 cells were transfected with siRNAs directed against gelsolin. Real-time PCR and Western blotting were used to determine the level of gelsolin. The ability of actin to inhibit DNase I activity was used to quantify monomeric and total actin level and calculate the state of actin polymerization. Fluorescence confocal microscopy was applied to observe gelsolin and vinculin distribution along with actin cytoskeleton organization.Key findingsIncreased level of gelsolin expression leads to its accumulation at the submembranous region of the cell accompanied by distinct changes in the state of actin polymerization and an increase in the migration of LS180 cells. In addition, LS180 cells overexpressing gelsolin form podosome-like structures as indicated by vinculin redistribution and its colocalization with gelsolin and actin. Downregulation of gelsolin expression in melanoma A375 cells significantly reduces their migratory potential.SignificanceOur experimental data indicate that alterations in the expression level of gelsolin and its subcellular distribution may be directly responsible for determining migration capacity of human cancer cells.     

Lumican affects actin cytoskeletal organization in human melanoma A375 cells

AIMS: Lumican, a small leucine-rich proteoglycan (SLRP), has attracted attention as a molecule of the extracellular matrix possibly involved in signalling pathways affecting cancer cell behaviour. The remodelling of the actin cytoskeleton, induced in response to external stimuli, is crucial for cell motility and intracellular signal transduction. The main goal of this study was to examine the effects of recombinant lumican on actin organization, the state of actin polymerization, actin isoform expression, and their sub-cellular distribution in the A375 human melanoma cell line. MAIN METHODS: Fluorescence and confocal microscopy were used to observe actin cytoskeletal organization and the sub-cellular distribution of cytoplasmic beta- and gamma-actins. The ability of actin to inhibit DNaseI activity was used to quantify actin. Western blotting and real-time PCR were used to determine the expression levels of the actin isoforms. KEY FINDINGS: A375 cells grown on lumican coatings changed in morphology and presented rearranged actin filament organization: from filaments evenly spread throughout the whole cell body to their condensed sub-membrane localization. In the presence of lumican, both actin isoforms were concentrated under the cellular membrane. A statistically significant increase in the total, filamentous, and monomeric actin pools was observed in A375 cells grown on lumican. SIGNIFICANCE: Novel biological effects of lumican, an extracellular matrix SLRP, on the actin pool and organization are identified, which may extend our understanding of the mechanism underlying the inhibitory effect of lumican on the migration of melanoma cells.     

Cofilin overexpression affects actin cytoskeleton organization and migration of human colon adenocarcinoma cells

The dynamic reorganization of actin cytoskeleton is regulated by a large number of actin-binding proteins. Among them, the interaction of ADF/cofilin with monomeric and filamentous actin is very important, since it severs actin filaments. It also positively influences actin treadmilling. The activity of ADF/cofilin is reversibly regulated by phosphorylation and dephosphorylation at Ser-3, with the phosphorylated form (P-cofilin) being inactive. Here, we studied the effects of overexpression of cofilin and two cofilin variants in the human colon adenocarcinoma LS180 cell line. We have generated the LS180 cells expressing three different cofilin variants: WT (wild type), Ser 3 Ala (S3A) (constitutively active) or Ser 3 Asp (S3D) (constitutively inactive cofilin). The cells expressing WT cofilin were characterized by abundant cell spreading and colocalization of cofilin with the submembranous F-actin. Similar effects were observed in cells expressing S3A cofilin. In contrast, LS180 cells expressing S3D cofilin remained longitudinal in morphology and cofilin was equally distributed within the cell body. Furthermore, the migration ability of LS180 cells expressing different cofilin mutants was analyzed. In comparison to control cells, we have noticed a significant, approximately fourfold increase in the migration factor value of cells overexpressing WT type cofilin. The overexpression of S3D cofilin resulted in an almost complete inhibition of cell motility. The estimation of actin pool in the cytosol of LS180 cells expressing S3A cofilin has shown a significantly lower level of total actin in reference to control cells. The opposite effect was observed in LS180 cells overexpressing S3D cofilin. In summary, the results of our experiments indicate that phosphorylation “status” of cofilin is a factor affecting the actin cytoskeleton organization and migration abilities of colon adenocarcinoma LS180 cells.     

Effect of overexpression of β- and γ-actin isoforms on actin cytoskeleton organization and migration of human colon cancer cells

Actins are eukaryotic proteins, which are involved in diverse cellular functions including muscle contraction, cell motility, adhesion and maintenance of cell shape. Cytoplasmic actin isoforms β and γ are ubiquitously expressed and essential for cell functioning. However, their unique contributions are not very well understood. The aim of this study was to determine the effect of β- and γ-actin overexpression on the migration capacity and actin cytoskeleton organization of human colon adenocarcinoma BE cells. In cells overexpressing β- or γ-actin, distinct cytoskeletal actin rearrangements were observed under the laser scanning confocal microscope. Overexpressed actins localized at the submembranous region of the cell body, especially near to the leading edge and on the tips of pseudopodia. The cells transfected with plasmids containing cDNA for β- or γ-actin were characterized by increased migration and invasion capacities. However, the migration velocity was statistically significantly higher only in the case of γ-actin overexpressing cells. In conclusion, the increased level of β- or γ-actin leads to actin cytoskeletal remodeling followed by an increase in migration and invasion capacities of human colon BE cells. These data suggest that expression of both actin isoforms has an impact on cancer cell motility, with the subtle predominance of γ-actin, and may influence invasiveness of human colon cancer.     

HER2/HER3 regulates extracellular acidification and cell migration through MTK1 (MEKK4)

Human MAP3K4 (MTK1) functions upstream of mitogen activated protein kinases (MAPKs). In this study we show MTK1 is required for human epidermal growth factor receptor 2/3 (HER2/HER3)-heregulin beta1 (HRG) induced cell migration in MCF-7 breast cancer cells. We demonstrate that HRG stimulation leads to association of MTK1 with activated HER3 in MCF-7 and T-47D breast cancer cells. Activated HER3 association with MTK1 is dependent on HER2 activation and is decreased by pre-treatment with the HER2 inhibitor, lapatinib. Moreover, we also identify the actin interacting region (AIR) on MTK1. Disruption of actin cytoskeletal polymerization with cytochalasin D inhibited HRG induced MTK1/HER3 association. Additionally, HRG stimulation leads to extracellular acidification that is independent of cellular proliferation. HRG induced extracellular acidification is significantly inhibited when MTK1 is knocked down in MCF-7 cells. Similarly, pre-treatment with lapatinib significantly decreased HRG induced extracellular acidification. Extracellular acidification is linked with cancer cell migration. We performed scratch assays that show HRG induced cell migration in MCF-7 cells. Knockdown of MTK1 significantly inhibited HRG induced cell migration. Furthermore, pre-treatment with lapatinib also significantly decreased cell migration. Cell migration is required for cancer cell metastasis, which is the major cause of cancer patient mortality. We identify MTK1 in the HER2/HER3-HRG mediated extracellular acidification and cell migration pathway in breast cancer cells.     

Gelsolin-independent podosome formation in dendritic cells

Podosomes, important structures for adhesion and extracellular matrix degradation, are claimed to be involved in cell migration. In addition, podosomes are also reported to be of importance in tissue remodelling, e.g., in osteoclast-mediated bone resorption. Podosomes are highly dynamic actin-filament scaffolds onto which proteins important for their function, such as matrix metallo-proteases and integrins, attach. The dynamics of the podosomes require the action of many proteins regulating actin assembly and disassembly. One such protein, gelsolin, which associates to podosomes, has been reported to be important for podosome formation and function in osteoclasts. However, podosome-like structures have been reported in gelsolin-deficient dendritic cells, but the identity of these structures was not confirmed, and their dynamics and function was not investigated. Like many other cells, dendritic cells of the immune system also form matrix degrading podosomes. In the present study, we show that dendritic cells form podosomes independently of gelsolin, that there are no major alterations in their dynamics of formation and disassembly, and that they exhibit matrix-degrading function. Furthermore, we found that gelsolin is not required for TLR4-induced podosome disassembly. Thus, the actin cytoskeleton of podosomes involved in dendritic cell extracellular matrix degradation appears to be regulated differently than the cytoskeleton in podosomes of osteoclasts mediating bone resorption.     

Transforming growth factor‐β1 modulates metalloproteinase‐2 and ‐9, nitric oxide,RhoA and α‐smooth muscle actin expression in colon adenocarcinoma cells

Colon carcinoma invasiveness is a process involving cell–cell and cell–matrix alterations, local proteolysis of the ECM (extracellular matrix) or changes in cytokine and growth factor levels. In order to evaluate the role of TGF‐β1 (transforming growth factor‐β1) and small G protein RhoA in tumour progression, the influence of TGF‐β1 treatment or RhoA‐associated kinase inhibitor on the production of NO (nitric oxide) and MMP‐2 and MMP‐9 (metalloproteinases‐2 and ‐9) was analysed in three human colon adenocarcinoma cell lines (HT29, LS180, SW948) representing different stages of tumour development. All the tested cell lines produced low amounts of MMP‐2 and MMP‐9. rhTGF‐β1 and the synthetic Rho kinase inhibitor (Y‐27632) decreased MMP‐2 secretion by colon cancer cells, especially in the most advanced stage of colon cancer. rhTGF‐β1 decreased NO secretion by cells, while Y‐27632 had no effect on it. Immunoblotting with anti‐RhoA antibodies followed by densitometry revealed that RhoA levels were slightly increased after incubation of colon carcinoma cells (SW948) with rhTGF‐β1. rhTGF‐β1 induced α‐smooth muscle actin (α‐SMA) expression, especially in high Duke's grade of colon cancer, while Y‐27632 blocked it. Summing up, in colon carcinoma cells, TGF‐β1 and RhoA protein may regulate tumour invasiveness measured as MMP, NO and α‐SMA expression or assayed using motility data and may be a good target for cancer therapy.     

Lumican – Derived Peptides Inhibit Melanoma Cell Growth and Migration

Lumican, a small leucine-rich proteoglycan of the extracellular matrix, presents potent anti-tumor properties. Previous works from our group showed that lumican inhibited melanoma cell migration and tumor growth in vitro and in vivo. Melanoma cells adhered to lumican, resulting in a remodeling of their actin cytoskeleton and preventing their migration. In addition, we identified a sequence of 17 amino acids within the lumican core protein, named lumcorin, which was able to inhibit cell chemotaxis and reproduce anti-migratory effect of lumican in vitro. The aim of the present study was to characterize the anti-tumor mechanism of action of lumcorin. Lumcorin significantly decreased the growth in monolayer and in soft agar of two melanoma cell lines – mice B16F1 and human SK-MEL-28 cells – in comparison to controls. Addition of lumcorin to serum free medium significantly inhibited spontaneous motility of these two melanoma cell lines. To characterize the mechanisms involved in the inhibition of cell migration by lumcorin, the status of the phosphorylation/dephosphorylation of proteins was examined. Inhibition of focal adhesion kinase phosphorylation was observed in presence of lumcorin. Since cancer cells have been shown to migrate and to invade by mechanisms that involve matrix metalloproteinases (MMPs), the expression and activity of MMPs were analyzed. Lumcorin induced an accumulation of an intermediate form of MMP-14 (~59kDa), and inhibited MMP-14 activity. Additionally, we identified a short, 10 amino acids peptide within lumcorin sequence, which was able to reproduce its anti-tumor effect on melanoma cells. This peptide may have potential pharmacological applications.     

Potentiation of cell migration by adhesion-dependent cooperative signals from the GTPase Rac and Raf kinase

The small GTPase Rac is thought to regulate cell movement by influencing actin cytoskeletal organization and membrane ruffling. However, cell migration also depends on the activation of mitogen-activated protein kinase (MAPK), which can regulate myosin motor function, an event critical for cell contraction. Evidence is provided that, during active cell adhesion to the extracellular matrix, Rac potentiates the MAPK pathway and influences cell migration by selectively synergizing with Raf kinase but not with Ras or MAPK kinase. In fact, the synergy between Rac and Raf kinase increases the chemotactic sensitivity of cells to epidermal growth factor by 1000-fold. Therefore, the role of Rac in cell migration not only depends on its ability to regulate actin cytoskeletal organization but also on its capacity to potentiate chemokine activation of MAPK in a manner that depends on active cell adhesion to the extracellular matrix.     

Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin

Osteoclast activation is important for bone remodeling and is altered in multiple bone disorders. This process requires cell adhesion and extensive actin cytoskeletal reorganization. Proline-rich tyrosine kinase 2 (PYK2), a major cell adhesion-activated tyrosine kinase in osteoclasts, plays an important role in regulating this event. The mechanisms by which PYK2 regulates actin cytoskeletal organization and osteoclastic activation remain largely unknown. In this paper, we provide evidence that PYK2 directly interacts with gelsolin, an actin binding, severing, and capping protein essential for osteoclastic actin cytoskeletal organization. The interaction is mediated via the focal adhesion-targeting domain of PYK2 and an LD motif in gelsolin's COOH terminus. PYK2 phosphorylates gelsolin at tyrosine residues and regulates gelsolin bioactivity, including decreasing gelsolin binding to actin monomer and increasing gelsolin binding to phosphatidylinositol lipids. In addition, PYK2 increases actin polymerization at the fibroblastic cell periphery. Finally, PYK2 interacts with gelsolin in osteoclasts, where PYK2 activation is required for the formation of actin rings. Together, our results suggest that PYK2 is a regulator of gelsolin, revealing a novel PYK2-gelsolin pathway in regulating actin cytoskeletal organization in multiple cells, including osteoclasts.     

Antiepileptic teratogen valproic acid (VPA) modulates organisation and dynamics of the actin cytoskeleton

The antiepileptic drug valproic acid (VPA) and teratogenic VPA analogues have been demonstrated to inhibit cell motility and affect cell morphology. We here show that disruption of microtubules or of microfilaments by exposure to nocodazole or cytochalasin D had different effects on morphology of control cells and cells treated with VPA, indicating that VPA affected the cytoskeletal determinants of cell morphology. Furthermore, VPA treatment induced an increase of F-actin, and of FAK, paxillin, vinculin, and phosphotyrosine in focal adhesion complexes. These changes were accompanied by increased adhesion of VPA-treated cells to the extracellular matrix. Treatment with an RGD-containing peptide reducing integrin binding to components of the extracellular matrix partially reverted the motility inhibition induced by VPA, indicating that altered adhesion contributed to, but was not the sole reason for the VPA mediated inhibition of motility. In addition it is shown that the actomyosin cytoskeleton of VPA-treated cells was capable of contraction upon exposure to ATP, indicating that the reduced motility of VPA-treated cells was not caused by an inhibition of actomyosin contraction. On the other hand, VPA caused a redistribution of the actin severing protein gelsolin, and left the cells unable to respond to treatment with a gelsolin-peptide known to reduce the amount of gelsolin bound to phosphatidylinositol bisphosphate (PIP2), leaving a larger amount of the protein in a potential actin binding state. These findings indicate that VPA affects cell morphology and motility through interference with the dynamics of the actin cytoskeleton.     

Subcellular distribution and expression of cofilin and ezrin in human colon adenocarcinoma cell lines with different metastatic potential

The dynamic reorganization of the actin cytoskeleton is regulated by a number of actin binding proteins (ABPs). Four human colon adenocarcinoma cell lines – parental and three selected sublines, which differ in motility and metastatic potential, were used to investigate the expression level and subcellular localization of selected ABPs. Our interest was focused on cofilin and ezrin. These proteins are essential for cell migration and adhesion. The data received for the three more motile adenocarcinoma sublines (EB3, 3LNLN, 5W) were compared with those obtained for the parental LS180 adenocarcinoma cells and fibroblastic NRK cells. Quantitative densitometric analysis and confocal fluorescence microscopy were used to examine the expression levels and subcellular distribution of the selected ABPs. Our data show distinct increase in the level of cofilin in adenocarcinoma cells accompanied by the reduction of inactive phosphorylated form of cofilin. In more motile cells, cofilin was accumulated at cellular periphery in co-localization with actin filaments. Furthemore, we indicated translocation of ezrin towards the cell periphery within more motile cells in comparison with NRK and parental adenocarcinoma cells.In summary, our data indicate the correlation between migration ability of selected human colon adenocarcinoma sublines and subcellular distribution as well as the level of cofilin and ezrin. Therefore these proteins might be essential for the higher migratory activity of invasive tumor cells.Key words: actin, cofilin, ezrin, colon adenocarcinoma.     

Effect of Lumican on the Migration of Human Mesenchymal Stem Cells and Endothelial Progenitor Cells: Involvement of Matrix Metalloproteinase-14

Background

Increasing number of evidence shows that soluble factors and extracellular matrix (ECM) components provide an optimal microenvironment controlling human bone marrow mesenchymal stem cell (MSC) functions. Successful in vivo administration of stem cells lies in their ability to migrate through ECM barriers and to differentiate along tissue-specific lineages, including endothelium. Lumican, a protein of the small leucine-rich proteoglycan (SLRP) family, was shown to impede cell migration and angiogenesis. The aim of the present study was to analyze the role of lumican in the control of MSC migration and transition to functional endothelial progenitor cell (EPC).

Methodology/Principal Findings

Lumican inhibited tube-like structures formation on Matrigel® by MSC, but not EPC. Since matrix metalloproteinases (MMPs), in particular MMP-14, play an important role in remodelling of ECM and enhancing cell migration, their expression and activity were investigated in the cells grown on different ECM substrata. Lumican down-regulated the MMP-14 expression and activity in MSC, but not in EPC. Lumican inhibited MSC, but not EPC migration and invasion. The inhibition of MSC migration and invasion by lumican was reversed by MMP-14 overexpression.

Conclusion/Significance

Altogether, our results suggest that lumican inhibits MSC tube-like structure formation and migration via mechanisms that involve a decrease of MMP-14 expression and activity.     

Patterning of the membrane cytoskeleton by the extracellular matrix

The extracellular matrices of different tissues contain components which affect the migration, morphology and differentiation of many types of cells. These forms of cell behavior often involve dramatic changes in cytoskeletal organization. Extracellular matrix components are recognized by specific cell surface receptors which span the membrane and interact with the actin cytoskeleton. In cultured cells, the matrix receptors are concentrated in sites of cell attachment called focal adhesions. Information that is conveyed from the extracellular matrix to the cytoskeleton may involve matrix components, cell surface receptors, as well as the proteins at the cytoplasmic face of the focal adhesion which link the receptors to the actin cytoskeleton.     

Identification of beta1 integrin as mediator of melanoma cell adhesion to lumican

Lumican is a small leucine-rich proteoglycan (SLRP) present in the dermal extracellular matrix. Previous data from our laboratory demonstrated that lumican decreases melanoma progression in vivo. Here, we show that melanoma cell migration is decreased by lumican and that this effect is due to an enhanced cell adhesion. The adhesion of A375 human melanoma cells on lumican was dose-dependent and required Mg²⁺ and Mn²⁺ divalent cations. Using a panel of monoclonal antibodies directed against integrin subunits, we showed that A375 cells can bind to recombinant lumican through β1 type integrins. Moreover, the use of rhodocetin, an inhibitor of α2 integrin, suggested that this particular subunit might also be involved in the interaction with lumican. The increased β1 integrin-mediated adhesion of melanoma cells to lumican might explain, at least in part, the anti-invasive effect of this SLRP.     

Actin in human colon adenocarcinoma cells with different metastatic potential

Four human colon adenocarcinoma cell line variants with different metastatic potential were used to examine whether a correlation exists between actin level, state of actin polymerization and invasiveness of tumour cells. Monomeric (G), total (T) and filamentous (F) actin were determined in the cytosolic fraction of these cells. A statistically significant decrease in G actin level and increase in the state of actin polymerization (measured by F:G actin ratio) were found in the cytosol of three cell variants with higher metastatic potential and invasiveness (EB3, 3LNLN, 5W) compared with the parental cell line (LS180). Our experimental data lead to the conclusion that there is a correlation between the metastatic capacity of human colon adenocarcinoma cells and the state of actin polymerization.     

Different phenotypes of colon carcinoma cells interacting with endothelial cells: role of E-selectin and ultrastructural data

Adhesion molecules are intimately involved in the process of tumour progression. Among them, E-selectin is an inducible endothelial cell adhesion molecule that plays a role in the interactions of neoplastic cells with the endothelium. These interactions are required for the trans-endothelial migration of tumour cells that leads to the growth at the new sites. Since the detailed events in the early phase of metastasis still remain poorly defined, our study has undertaken an electron-microscopic analysis of the interactions of human colon carcinoma cells with endothelial cells as well as an analysis of the effect of recombinant purified E-selectin in the cell signalling involved in colon cancer cell malignant phenotype. Results revealed that SW480 and T84 colon cancer cell lines show different features, different adhesion kinetics, a different cytoskeletal organization, and a different tyrosine phosphorylation pattern when seeded on an endothelial cell monolayer or recombinant E-selectin. In particular T84 cancer cells adhere more efficiently to the E-selectin and this interaction is associated with pronounced morphological changes, actin redistribution and filopodial processes, and an increase in tyrosine phosphorylation of different proteins. These data support the hypothesis that E-selectin ligand is not only a cell-cell adhesion molecule but also initiates a signalling transduction pathway inside the cells.     

Lumican inhibits cell migration through α2β1 integrin

Lumican, an extracellular matrix protein of the small leucine-rich proteoglycan family, has been shown to impede melanoma progression by inhibiting cell migration. In the present study, we show that lumican targets α2β1 integrin thereby inhibiting cell migration. A375 melanoma cells were transfected with siRNA directed against the α2 integrin subunit. Compared to A375 control cells, the anti-migratory effect of lumican was abrogated on transfected A375 cells. Moreover, lumican inhibited the chemotactic migration of Chinese hamster ovary (CHO) cells stably transfected with α2 integrin subunit (CHO-A2) but not that of wild-type CHO cells (CHO-WT) lacking this subunit. In contrast to CHO-WT cells, we observed in time-lapse microscopy a decrease of CHO-A2 cell migration speed in presence of lumican. Focal adhesion kinase phosphorylated at tyrosine-397 (pFAK) and total FAK were analysed in CHO-WT and CHO-A2 cells. A significant decrease of the ratio pFAK/FAK was shown in presence of recombinant human lumican. Using solid phase assays, a direct binding between lumican and the α2β1 integrin was demonstrated. This interaction did not involve the glycan moiety of lumican and was cation independent. Lumican was also able to bind the activated I domain of the α2 integrin subunit with a K_d ≥ 200 nM. In conclusion, we demonstrated for the first time that the inhibition of cell migration by lumican depends on a direct binding between the core protein of lumican and the α2β1 integrin.     

Actin-associated protein palladin promotes tumor cell invasion by linking extracellular matrix degradation to cell cytoskeleton

Basal-like breast carcinomas, characterized by unfavorable prognosis and frequent metastases, are associated with epithelial-to-mesenchymal transition. During this process, cancer cells undergo cytoskeletal reorganization and up-regulate membrane-type 1 matrix metalloproteinase (MT1-MMP; MMP14), which functions in actin-based pseudopods to drive invasion by extracellular matrix degradation. However, the mechanisms that couple matrix proteolysis to the actin cytoskeleton in cell invasion have remained unclear. On the basis of a yeast two-hybrid screen for the MT1-MMP cytoplasmic tail-binding proteins, we identify here a novel Src-regulated protein interaction between the dynamic cytoskeletal scaffold protein palladin and MT1-MMP. These proteins were coexpressed in invasive human basal-like breast carcinomas and corresponding cell lines, where they were associated in the same matrix contacting and degrading membrane complexes. The silencing and overexpression of the 90-kDa palladin isoform revealed the functional importance of the interaction with MT1-MMP in pericellular matrix degradation and mesenchymal tumor cell invasion, whereas in MT1-MMP–negative cells, palladin overexpression was insufficient for invasion. Moreover, this invasion was inhibited in a dominant-negative manner by an immunoglobulin domain–containing palladin fragment lacking the dynamic scaffold and Src-binding domains. These results identify a novel protein interaction that links matrix degradation to cytoskeletal dynamics and migration signaling in mesenchymal cell invasion.     

PXR基因外显子3甲基化与肠癌细胞对5氟尿嘧啶的耐药性相关

孕烷X受体（pregnane X receptor, PXR）可通过调节细胞色素P450同工酶3A4 -CYP3A4的表达而影响肿瘤细胞对化疗的敏感性,而其表达水平则会受到自身基因 甲基化的影响.本文研究了结肠癌组织中pxr基因甲基化的分布情况及其对pxr, cyp3a4表达的影响,并在多种结肠癌细胞系中分析了pxr基因甲基化是否与5-氟尿嘧 啶 (5-FU)耐药性相关.收集结肠癌病灶区、癌旁区及正常结肠组织样本,分别提取基因组DNA及RNA.PCR限制性酶切分析检测pxr基因外显子3甲基化;real-time PCR检测pxr及cyp3a4基因的表达.鉴定LOVO、LS180、LS174T、HT29、HCT116等5种结 肠癌细胞中pxr外显子3甲基化与pxr, cyp3a4表达的相关性并分别筛选出PXR高/低表达的细胞株进行5-FU耐药性分析.结果显示,结肠癌病灶组织中pxr外显子3甲基化频率显著增加,伴有pxr,cyp3a4表达的增强.在结肠组织及结肠癌细胞系中,pxr与cyp3a4的表达均密切相关,且均与pxr甲基化程度相关.PXR高表达细胞株LS180对5-FU的耐药性显著升高,以siRNA分别下调pxr及cyp3a4的表达,均可增加LS180对5 -FU的敏感性.结果提示,pxr基因外显子3区甲基化与PXR及CYP3A4的高表达密切相关,并与结肠癌细胞对5-FU的抗药性相关.