Inhibitory Effect Of Doxycycline On The Adhesion Of Melanoma Cells And Its Molecular Mechanisms

Objective:To investigate the inhibitory effect of Doxycycline on the adhesion of melanoma cells and its mechanism in malignant melanoma model by using MMT, cell adhesion experiment, flow cytometry(FCM), immunofluorescent staining,Western blot methods and tumor-bearing mice model. To investigate the effect and its mechanism of Doxycycline on malignant melanoma, and provide a basis for further considering tetracycline-based antibiotics for use in anti-tumor treatment.Methods:Melanoma B16F110, WM351 and WM451 cell lines were treated with Doxycycline at different doses and through different administration methods. The Pro-group, drug is administered immediately after the cells were seeded, namely prior to cell attachment. The after-group, drug is administered 24h after the cells were seeded, namedly after cell attachment. Cells that were placed in solution without doxycycline were assigned as the control group. The differences between the cytotoxic effect of pro-adherence and after-adherence were observed. To investigate the effect of adherence inhibition in melanoma cells by doxycycline, three cell-lines were used-C57BL mouse orgin B16F10 cells, human melanoma cells WM35, and WM35 lung metastasis cell-line WM451. The differences of inhibitory effect of doxycycline on these cells were observed. To investigate whether the adhesion-inhibited cells underwent apoptosis, the B16F10 cells were treated with doxycycline. To assess apoptosis, cell death detection Annexin V-PI assay by FCM was used. Doxycycline also presents its effects on the cytoskeleton by destroying the construction of actins and microtube in melanoma cells. Immunofluorescent staining was done on the B16F10 cells treated with doxycycline. Western blot were used to observe the different levels of FAK and its phosphorylation on these cells. To further determine whether FAK and its phosphorylation were also significant while doxycycline was present in vivo, a mouse melanoma tumor model, the B16 cell-line was chosen. A total of 20 mice were randomly divided into the treatment group(10 mice) and the control group(10 mice). Eight days after the tumor cells were engrafterd, the tumor masses were detected. At this time, doxycycline solution was administered intraperitoneally in the treatment group. The mice in the control group received an equivalent of 0.9% NaCl solution as placebo. The mice were sacrificed by cervical decapitation 3 days following continuous treatment. A part of the tumor without necrosis was also collected and detected by Western blot.Results:MTT analysis showed that the inhibition ratio was significantly higher in the group receiving treatment before cell adhesion than in the group receiving treatment after cell adhesion (P<0.05), and cell morphology exhibited suspension and fragmentation. This phenomenon shows that cell adhesion inhibition may be an important process in the function of doxycycline. Cell adhesion experiment showed that Doxycycline had an inhibitory effect on the adhesion of B16F110, WM351 and WM451 cells, with a statistical significance (P<0.05). An 80% reduction of the cells was evident after treatment with doxycycline concentration of 0.1 ug/mL for 2h compared to solvent control. The WM451 cells showed less adherence inhibition than the WM35 and B16F10 cells. To assess apoptosis, cell death detection Annexin V-PI assay by FCM was used. The results show an increase in the number of cells in pro-apoptosis among the B16F10 cells. The percentage of pro-apoptosis increased ninefold in the control group. Immuno-fluorescent staining forβ-actin anda-tubulin was done on the B16F10 cells treated with 1 ug/ml and 10 ug/ml of doxycycline for 12 h. The first indication was that doxycycline influenced the dispersion of the micro-tube constituted by a-tubulin and the accumulation ofβ-actin in 1 ug/ml. The cells shrank and were suspended in 10 ug/ml of doxycycline; chromatin con-densation in the nuclei was also detected. Western blot revealed that FAK expression in the three cell lines was decreased at 12h after Doxycycline treatment. The results showthat the levels of FAK-Tyrosine397 phosphorylation were significantly down-regulated after treatment with doxycycline in these three cell-lines. FAK phosphorylation decreased by 80% at 1 ug/ml of doxycycline after comparison with the control group. Observations from the mice melanoma model suggested obvious changes in FAK expression and its phosphorylation level (P<0.05). The treatment group exhibited about twofold of FAK expression and phosphorylation compared to the control group.Conclusion:Doxycycline can interfere the adhesion of melanoma cells, possibly through inhibiting FAK expression and its phosphorylation. These data provide a basis for further considering tetracycline-based antibiotics for use in anti-tumor treatment.