Morphological Observation And Relative Roles Of MMP-2 And TIMP-2 In C6 Cells With Highly Invasive Ability

Glioma, the most common malignant brain tumor of adults is characterized by aggressive tumor cell invasion into the surrounding brain, making complete surgical excision nearly impossible. Glioma invasion is believed to involve a complex series of integrated events consisting of tumor cell adhesion, extracellular matrix (ECM) proteolysis, and cell migration within the surrounding micro-environment.C6 cells derived from rat brain glioma cells induced by N - nitrosomethyl-urea, which is in common use as a kind of glioma cells. As usually, C6 cells is a good model in the glioma investigation, because it's pathological character is coincident with human glioblastoma's.Further elucidation of MMP and TIMP regulatory mechanisms and their roles during tumor progression is critical especially in view of the disappointing results of recent clinical trials with MMP inhibitors. The goal of this study was to identify specific mechanisms contributing to glioma invasion by isolating a sub-population of more invasive cells from the glioma cell line C6 and comparing their phenotypes for relevant differences.Section A Morphological observation of C6 cells with highly invasive ability selected through Transwell chambersObjectiveIsolating a subpopulation of more invasive cells from the glioma cell line C6and comparing their phenotypes for relevant differences, to detect the tumor biological characteristic relation to glioma invasion.Method1. Selection of the highly invasive C6 cellsSix -well Transwell polycarbonate membrane inserts with 8. 0 - μm pores (Costar, USA) were coated from the bottom with 10μg/ml collagen type I. To generate C6 - C₁ cells, the first 5% of invading cells from C6 were selected u-sing the same collagen - coated Transwell filters under serum - free conditions. To obtain C6 - C_Res cells, noninvading cells on the upper surfaces of the filters were harvested in a similar fashion and cultured.2. Transwell Invasion AssayFilters were then washed, and cells on the upper surface were removed with cotton swabs. Cells that had invaded and adhered to the lower surface were fixed in 4% paraformaldehyde for 10 min and stained with 0. 25% crystal violet. Eight random fields were counted to determine the number of cells invaded.3. Wound AssayConfluent monolayers were scratched with a plastic pipet tip to create a uniform , cell - free wound area that was then inspected at regular time intervals. At each time point eight photographs of each wound were taken and the distance between the opposing edges was measured at two points on each photograph. The distance migrated in micrometers was calculated as the difference of the scratch width at the beginning of the assay and that at each indicated time point.4. Actin ImmunofluorescenceTo observe the cytoskeletal features of motility of C6 - C₁ cells.Results1. There were no clearly discernable differences in cell morphology between the parental C6, C6 - C_Res, and C6 - C₁ under phase - contrast microscopy.2. The selected C6 - C₁ cells exhibit increased invasiveness and motility,respectively (180. 6 ±20. 8) and (62. 4 ± 13. 2) , which increased three - fold compared to parental C6 cells, indicating difference significantly ( p <0.01 ).3. The migration of C6 - C₁ cells at 3h, 5h, 8h, respectively (172.0 ± 19. 1),(357.2± -20.3)and(469.8±13.2),is much faster than C6-C_Res at 3h (p <0. 05) , and differences significantly in mobility at 5h, 8h were assessed compared to parental C6 and C6 - C_Res cells ( p <0.01 ).4. C6 - C₁ cells display cytoskeletal alterations of morphology associated with tumor cell invasion.Conclusions1. We firstly isolated the highly invasive C6 - C₁ cells through Trandwell chambers and observed the morphological characteristic associated with tumor cell invasion.2. The selected C6 - C₁ cells exhibit increased invasiveness and motility compared to parental C6 cells, indicating differences significantly ( p <0. 001 ).3. C6 -C₁ cells feature cytoskeletal rearrangements that correlate with their enhanced invasiveness.Section B Function of MMP - 2 in highly invasive C6 cellsObjectiveTo study the function of matrix metalloproteinase - 2 ( MMP - 2) in the highly invasive C6 cells, further elucidate their regulatory mechanisms and roles during tumor progression.Methods1. ZymographyGelatinolytic activity of conditioned media was detected by gelatin zymography. Serum - free media conditioned for 48h were subjected to SDS - PAGE u-sing 10% acrylamide gels containing 0. 1% gelatin. The gels were then incubated in 2. 5% Triton X - 100, for 16h at 37℃. Gels were then stained with Coo-massie Brilliant Blue R250 for 4h and briefly destained in destaining solution for 1 -2h. Gelatinolytic activity was detected as transparent bands on a blue background.2. Western blotConfluent cells were homogenized in 200μL lysis buffer, then centrifuged (4℃,16000 r/min) for 2h to pellet cell debris. Equal amounts of protein in sample buffer were loaded, SDS - PAGE was performed. After transfer to nitrocellulose membranes, samples were blocked in TTBS with 5 % BSA and incubated with primary antibodies at 4℃ overnight. Blots were incubated with secondary antibodies for 2h at room temperature, band were visualized.Results1. Zymographic analysis: There was an 80% increase in both 64 kDa intermediate and 62 kDa fully activated MMP - 2 processed forms compared to parental and residual cells, which had comparable levels of MMP - 2 activation ( p < 0.0001).2. Western blots analysis on cell lysates revealed no detectable changes in MT1 - MMP levels between C6 - C₁ and C6 cells.ConclusionsThe increase levels of MMP2 activation was associated with ability of glioma invasion and metastasis.Section C Expression of TIMP -2 in C6 cellswith highly invasive abilityObjectiveTo identify tissue inhibitors of metalloproteinase - 2 ( TIMP - 2 ) releasedby the highly invasive C6 cells selected through transwell chambers and to detect the roles of matrix metalloproteinase - 2 ( MMP - 2 ) and TTMP - 2 in glioma invasion and motility.Methods1. ZymographyGelatinolytic activity of conditioned media was detected by gelatin zymography. Serum — free media conditioned for 48h were subjected to SDS — PAGE u-sing 10% acrylamide gels containing 0. 1% gelatin. The gels were then incubated in 2.5% Triton X - 100, for 16h at 37℃. Gels were then stained with Coo-massie Brilliant Blue R250 for 4h and briefly destained in destaining solution for 1-2h. Gelatinolytic activity was detected as transparent bands on a blue background.Reverse zymography was performed the same way, except that samples of media were run in 15% acrylamide gels containing 0. 225% gelatin and 50 ng/ ml purified MMP-2.2. Western blotConfluent cells were homogenized in 200μL lysis buffer, then centrifuged (4℃, 16000 r/min) for 2h to pellet cell debris. Equal amounts of protein in sample buffer were loaded, SDS -PAGE was performed. After transfer to nitrocellulose membranes, samples were blocked in TTBS with 5% BSA and incubated with primary antibodies at 4℃ overnight. Blots were incubated with secondary antibodies for 2h at room temperature, band were visualized.3. Semi quantitative RT - PCRAccording to TRIzol Reagent kit. The mRNA amounts of TIMP - 2 were measured by reverse transcript polymerase chain reaction ( RT - PCR ) and normalized to the mRNA levels of GAPDH. The PCR program used as following: 94℃ strand denaturation for 4 min, followed by a 3 step cycling program of 94℃ for 40 secs, annealing at 56℃ for 40 seconds, and extension for a total of 20, 25, 35cycles. The last cycle was followed by a 2 min extension at 72℃. The products were then loaded onto agarose gels, stained with ethidium bromide,...