Research Of A Novel Retinoic Acid Derivative4-amino-2-trifluoromethyl-phenyl Retinate (ATPR) On Induced Differentiation Of Human Glioma Cell U87

Objective Glioma, an aggressive and prevalent brain tumor, is considered to be one of the most severe forms of human cancer consisting35.26%-60.96%of the primary intracranial malignant tumor.The treatment of malignant gliomas is currently one of the most difficult challenges in oncology.Although the progress of surgery, radiotherapy, chemotherapy and other treatment techniques in recent decades have made improvement of prognosis in glioma patients, while it unfortunately has not increased the overall survival yet. It is imperative to find an effective treatment to control this malignant disease. In recent years, the concept of drug-induced differentiation therapy which induces tumor cells differentiate into more mature cells rather than directly killing or eliminating tumor cells as traditional treatments strategy has drawn wide attention. A variety of differentiation inducer has been found recent years.4-amino-2-trifluoromethyl-phenyl retinate (ATPR) is one of the recently synthesized all-trans retinoic acid derivatives with good effect to induce tumor cell differentiation. In this study, we observed the effect of ATPR on the U87glioma cells and detected its role on cell proliferation, invasion,induction of differentiation and promotion of apoptosis. Also we aim to provide experimental evidence for the treatment of gliomas and find appropriate differentiation inducer.Methods U87cells were divided into blank control group, solvent control group, positive control group and the experimental group. After treatment with PRMI-1640cell culture medium, PRMI-1640cell culture medium containing dimethyl sulfoxide(DMSO),2mmol/L of sodium phenylbutyrate(SPB) and different concentrations of ATPR (final concentration was1×10-5,1×10-6,1×10-7,1×10-8and1×10-9mol/L respectively) respectively, U87cells were analysised to different techniques. Cell Counting Kit8(CCK-8) and colony formation assay were adapted to analysis the cell proliferation of U87; Optical microscopy and electron microscopy were used to observe the cell morphology change; Tunnel test was performed to exhibit the cell apoptosis; Transwell test was taken to show the cell invasiveness; Western blotting was carried to show the expression of glial fibrillary acidic protein (GFAP); Flow cytometry was utilised to exhibit cell cycle distribution.Results.1、ATPR inhibit the cell proliferation of U87cells.1.1、Cell growth curve showed DMSO group and blank group cells grow thrive while SPB group and each dose of ATPR group could inhibit the cell proliferation of U87, The significant inhibitory effect came72h after administration. Furthermore, with the ATPR concentration increasing, the degree of inhibition is more obvious, suggesting a dose-related inhibitory effect on U87cells. The IC50for72h is1.75×10-5mol/L.1.2、Colony formation assay showed cells in SPB group and1×10-5,1×10-6mol/L ATPR groups grow scattere around and fail to form an effective clone. Cloning:. efficiency of cells in1×10-7,1×10-8,1×10-9mol/L ATPR group is (4.97±1.22)%,(17.65±5.17)%,(26.24±3.65)%respectively, lower than that in the control group (43.26±8.81)%. The difference is statistically significant (t=7.458,4.341,3.091, P <0.05).No significant difference of cloning efficiency was found between DMSO group (33.26±5.40)%and the control group (t=1.676, P>0.05).2、ATPR down-regulate the invasiveness of U87cells. The invasiveness of U87cells decreased after72hours’ treatment of ATPR. Transwell test shows the invasion capacity of cells in SPB group and ATPR groups with a consentration of1×10-5,1×10-6,1×10-7and1×10-8mol/L decreased. There is significant difference compared with the control group (P<0.05). No significant difference between1×10-9mol/L ATPR and DMSO group, suggesting ATPR can decrease the invasiveness of U87cells.3、ATPR promotes the apoptosis of U87cells. The proportion of apoptotic cells increased after72h of ATPR treatment. Tunnel Experimental resulted that, the number of apoptotic cells in SPB group and ATPR groups with the consentration of1×10-5,1×10-6,1×10-7and1×10-8mol/L increased obviously. Furthermore the apoptosis trend more significant with the increasing of ATPR consentration. It showed a significant difference when compaired to the control group (P<0.05). While no significant difference has been found between DMSO group,1×10-9mol/L ATPR group and the control group (P>0.05), indicating ATPR can promote apoptosis of U87cells.4、ATPR trigger cell cycle arrest of U87cells.the proportion of cells in G1/G0phase increased in SPB group and ATPR groups with different consentration of1×10-5,1×10-6and1×10-7mol/L compared with the control group(t=8.339,5.942,4.473,2.864, P<0.05). And the proportion of cells in S phase which represents the DNA synthesis decreased significantly (t=9.946,7.209,4.627,2.868, P<0.05) which indicating that ATPR can exert disfunction of cell cycle progression and cause cell arrest in G1/G0phase, in which reduces the proportion of cells in S phase.5、ATPR change the cell morphology of U87cells. Optical microscope observation showed cell morphology of U87cells in the control group and DMSO group is similar with large cell volume and nuclei, high nuclear/cytoplasmic ratio, less cell processes. After ATPR treatment, U87cells showed morphological maturation, showing reduced cell size and cell nuclei, increased projections and decreased nuclear/cytoplasmic ratio. The morphological maturation tends to be more obvious with the increasing of ATPR consentration. However, cell morphology in1×10-6mol/L ATPR group is similar to that in SPB group, and the degree of maturity seems more exciting than that in1×10-5mol/L ATPR group. Electron microscopy showed those cells in control group and DMSO group with larger nuclei, fewer organelles, while cell size and cell nucleus of cells in SPB group and ATPR group are smaller. Cell processes increased in varying degrees, and abundant organelles including a lot of mitochondria and endoplasmic reticulum seen obviously. A lot of swelling of mitochondria were showed in1×10-5mol/L ATPR group.6、ATPR up-regulates the GFAP expression of U87cells. Western blot indicated the high expression of GFAP in cells of SPB group and high concentration ATPR groups. It performanced deepened and widened strip. It resulted that the expression level of GFAP in SPB group and ATPR groups with different consentration of1×10-5,1×10-6,1×10-7and1×10-8mol/L is higher than that in the control group. No obvious difference was found between1×10-9mol/L group and the control group.Conclusion:1、ATPR can effectively inhibit the proliferation, promote apoptosis and reduce the invasiveness of U87cells;2、ATPR is able to exert the cell cycle arrest, increasing GFAP expression and promoting morphological maturation of U87cells;3、ATPR is expected to become a suitable tumor differentiation induce agent and bring new hope for the clinical treatment of glioma.