| Objective: To examine the inhibition of doxycycline on the microcirculation patterns and proliferative activity of melanoma in mouse melanoma transplanted tumor model. The effects and primary mechanism of doxycycline on melanoma growth would be elucidated. These results would be hoped to elicit a novel approach to the clinical treatment of melanoma.Methods: Forty-seven C57 mice were randomly divided into doxycycline treatment group and control group. The melanoma transplanted model was established by subcutaneous injection B16 cells suspending liquid in mouse. Mice in treatment group were injected with doxycycline and mice in control group with physiological saline solution everday when tumor could be touched. Seven days later, ten mice in treatment group and ten mice in control group were killed, and fourteen days later all other mice were killed. These tumor were peeled off and weighted, then small parts of tumor were cut from tumor and stored in -80°C refrigerator. These residual tumor were cut and routinely fixed with formalin. The morphological characteristics of slides of tumor specimens stained with HE and PAS were examined in microscope. The expression level of MMP-2, MMP-9, VEGF, PCNA were examined by IHC. Methods of counting: ten representative high visual fields were selected in each slide avoiding of putrescence areas, and the number of positive staining tumor cells werecounted in 100 tumor cells in each field. Staining index was used to evaluate the entire staining status of each slide. The activity of MMP-2, MMP-9 between different groups was examined by gelatin zymography. Counting methods of microcirculation patterns: endothelial-dependent vessels and VM were counted in ten high fields in each slide combined with PAS staining. SPSS statistic software was used to deal with data and the boundary of statistical significance was set at P<0.05. Results: Growth rate of tumor in treatment group were slower than control group during the experiment. The IR (inhibition rate) was 52.36% after 7 days treatment, but the IR was decreased to 35.63% afer 14 days treatment. By gelatin zymography, the enzyme activity of MMP-9, actived-MMP-2 and MMP-2/pro-MMP-2 in treatment group were lower than control group (P<0.01), but the enzyme activity of pro-MMP-2 between two groups was not significant different (P>0.05). After 7 days treatment, the expression level of MMP-2 and MMP-9 in treatment group was lower than control group (P<0.01, P<0.05) and the expression level of VEGF in treatment group was higher than control group (PO.05), the density of endothelial-dependent vessel and VM in treatment group were lower than control group (P<0.01, P<0.05). Afer 14 days treatment, the expression level of MMP-2 and MMP-9 in treatment group was lower than control group (P<0.01, P<0.01) and the expression level of VEGF in treatment group was higher than control group (P<0.05), the density of endothelial-dependent vessel in treatment group were lower than control group (P<0.01), but the density of VM between two group was not different (P>0.05). In addition, these was no different expression level of PCNA between two groups afer 7 days treatment (P>0.05), but the expression level of PCNA in treatment group was lower than control group after 14 days treatment. Conclusion: Doxycycline could inhibit the growth of malignant melanoma, therewere two possible mechanisms: (1) It could inhibit expression and activity of MMP-2 and MMP-9. In early stage of malanoma, endothelial-dependent vessel and VM were inhibited by doxycycline and inhibiton effect of doxycycline on tumor was significant. But in later stage, only the endothelial-dependent vessel, not VM was inhibited by doxycycline. The inhibiting effect of doxycycline on tumor was decreased. Higher hypoxia tension in later stage was speculated to allow embryonic-like melanoma cells activing MMP-2-independent signal transduction pathway to promote VM formation and enhance tumor cells's plasity. The conclusion that inhibiting effect of doxycycline on tumor was reduced because VM was not effectively inhibited could explain the unsatisfying effects of anti-angiogenesis therapy only targeted the endothelial vessel. (2) It could affect the tumor cell cycle after a period of time. Proliferative activity of melanoma was not affected afer 7 days treatment, but decreased afer 14 days treatment.
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