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Inhibitory Effect of Doxycycline on the Adhesion of Melanoma Cells and Its Molecular Mechanisms

Author: DongHengLei
Tutor: SunBaoCun
School: Tianjin Medical University
Course: Pathology and Pathophysiology
Keywords: Doxycycline Melanoma Cell adhesion Focal adhesion kinase Anti-tumor effects
CLC: R965
Type: Master's thesis
Year: 2011
Downloads: 24
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Abstract

Objective: high hematogenous metastasis of malignant tumors - melanoma research model, using the cytotoxicity assay, adhesion assay, flow cytometry, immunofluorescence staining, Western Blot and tumor-bearing animal models such as technology, research doxycycline on inhibition of melanoma cell adhesion and molecular mechanisms. Preliminary clarify the effect and mechanism of drug for malignant melanoma, and thus provide experimental evidence for treatment of new ways for exploring malignant melanoma. Method: Department of melanoma cells B16F110 WM351 WM451 respectively before and after the adherent treatment administered in accordance with the drug concentration gradient by MTT were divided into two modes of administration, that immediately decking after administration of pre-treatment (adherent) and cultured for 24h after the administration of post-treatment (adherent), drug-free saline as solvent control group, observed data given doxycycline treatment differences adherent and adherent. To further evaluate the efficacy and quantify cell adhesion experiment of three melanoma cell concept Cha Duoxi doxycycline inhibition of adhesion and differences of the three types of cells. To further validate doxycycline treatment whether early apoptosis cell interference polarization doxycycline induced melanoma cell anoikis AnnexinV-PI-based flow cytometry analysis. The cytoskeleton changes after hormone treatment by the immunofluorescence staining concept Cha Duoxi ring. Use of Western Blot Detection of FAK expression and phosphorylation of the three cell level changes. Establish tumor models using C57/BL mouse, 20 mice were randomly grouped into two groups, namely the treatment group and the control group. 8d after transplantation site palpable tumor, doxycycline intraperitoneal injection at this time, the control group was injected with the same dose of saline. 3d after administration, the mice were sacrificed to obtain tumor tissue by Western Blot FAK phosphorylation levels of change. RESULTS: MTT experimental results show that the cell inhibition rate of adherent dosing significantly higher than the adherent dosing group, mainly on the cell morphology suspended spherical, the phenomenon suggests that doxycycline may tumor impact on the role of cell adhesion. Cell adhesion test results showed that doxycycline these three melanoma cells have adhesion inhibition incubated 0.1μg/mL for 6h cell adhesion rate decreased by 80% compared with the control group, doxycycline WM451 cell adhesion inhibition less in the other two types of cells. AnnexinV-PI flow cytometry analysis of doxycycline induced melanoma cell anoikis, the results show the proportion of early apoptotic B16F10 cells with multi-1μg/mL doxycycline treatment 2h increased about 9 times . The microfilament protein tubulin staining by immunofluorescence staining of melanoma B16F10 found: 1μg/mL 10μg/mL multi doxycycline treatment cells after 12 h, can be seen microtubule depolymerization, dissolved and microfilament abnormal accumulation of concentrated cell shrinkage and nuclear chromatin aggregation under the high concentration of multi doxycycline treatment. Western Blot results show that the use of doxycycline treatment after 12h, B16F10, WM351 and WM451 three cell showed decreased FAK expression levels of the different levels of tyrosine 397 position on the extent of phosphorylation of FAK in significantly reducing drug concentration 1μg/mL FAK phosphorylation levels of three cell lines was reduced by more than 80%. Experimental mouse tumor models, the third day when the administration that the animals were killed to obtain tumor tissue for the detection of FAK phosphorylation results show that FAK phosphorylation levels were significant changes administration group than in the control group to reduce less than 50% (P lt; 0.05). Conclusion: doxycycline may inhibit FAK phosphorylation of melanoma cells, which interfere with the adhesion of tumor cells, and then play the anti-tumor effect. The molecular mechanisms proposed will contribute to the further evaluation of multi-clinical anti-tumor effect of doxycycline, also laid the foundation to clarify the depth molecular mechanism.

Inhibitory Effect of Doxycycline on the Adhesion of Melanoma Cells and Its Molecular Mechanisms

Abstract

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