The Study Of The Effect And Mechanism Of Doxycycline On The Lung Cancer Cells

With the further understand of MMPs, people take amphasis on the effects of MMPs to the growth, invision and metastasis of tumour. Using MMPs inhibitor for oncotherapy has been a new trend at present. Doxycycline(DOX), a semisynthesis tetracyclines antibiotic, has been studied widespreadly because of its long half-life and little toxicity.Objective To investgate the effects of DOX on the proliferation and metastatic potential of human adenocarcinoma of lung A549 cells and their relationship with the expression of MMPs , VEGF and AP1, demonstrate the mechanism of treating lung cancer using DOX at cellular and molecular levels .Method The effects of DOX on the proliferation of the A549 cells were observed by MTT assays. Electron microscope, fluorescent microsopy, DNA ladders and flow cytometry were used to observe the effects of DOX on apoptosis of A549. The effects of DOX on adhesion, invasion and motility potential of human adenocarcinoma of lung carcinoma A549 cells were investigated with cell attachment assay on Matrigel, wound assay, invasive assay, tumor metastasis experiment in nude mice. Moreover, Gelatin zymography, RT-PCR and western Blot were used to detect the effects of DOX on the expression of MMP2. Immunohistochemistry assay were used to detect the expression of VEGF in A549 cells and the samples of the metastastic foci sectioned from the surfaces of nude mouse lungs. RT-PCR and western Blot were used to detect the effects of DOX on the expression of c-fos and c-jun.Results1. Doxycycline more than 5 mg/L inhibit the proliferation of A549 in a nose and time dependent manners.2. After the DOX ( 20, 40 mg/L) treatment, the results of Electron microscope, fluorescent microsopy showed that the apoptotic sign of A549 cells, that is the volume of cells were decreased, nuclear contraction, chromation condensation and apoptotic bodies formed. Agrose gel electrophoresis of DNA fragements showed a ladder-like patten. The results of Annexin V- PI showed that the apoptotic rate were 2.22 %,5.86 %,12.6 %,20.59 % respectively with the treatment of DOX (0,10,20,40 mg/L).3. Attachment rates of A549 cells treated with DOX at 2.5, 5, 10 mg/L were 81.6 %, 67.2 % and 55.3 %respectively, whereas that of the control was 96%. The motility of A549 cells on Matrigel was decreased by DOX at 2.5, 5, 10 mg/L , the inhibitory rate were 3.7 %, 10.8 % and 18.2 %respectively for 24 h, 5.8 %, 20.2 % and 35.8 % for 48 h. DOX at 5, 10 mg/L can inhibit invasive potential of A549 cells, the inhibitory rate of invasion were 12 % and 33 % respectively, while DOX at 2.5 mg/L has no this effect. The number of metastatic foci on the lung surfaces in the nude mice treated with DOX at 30mg/kg/day and that in the controls were 74±19 and 15±35 respectively.4. The activity of 72 kDa matrix metalloproteinase (MMP2) of A549 cells was decreased at different levels by 2.5, 5 and 10 mg/L of DOX, the most decreasment was observed at 10 mg/L. The expression of MMP2 was inhibited by 10 mg/L of DOX.5. In vitro, the VEGF expression was significantly decreased in A549 cells which had been treated with DOX at 10 mg/L of DOX for 48h than in the controls. Consisting with the assay in vitro, the lower VEGF expression was also detected in the samples of foci in the DOX- treated nude mice.6. In vitro, the c-jun expression was significantly decreased in A549 cells which had been treated with DOX at 10 mg/L of DOX for 48 h than in the controls.Conclusion:1. Pharmacological concentration of DOX can inhibit the proliferation of A549 cells in vitro while large dose of DOX can induce the apoptosis of A549 cells.2. Pharmacological concentration of DOX can inhibit the adhension and motility of A549 cells to Matrigel, decrease invasion to Matrigel , decrease the metastatic foci of A549 cells in lungs of nude mice treated with of DOX at safe dose.3. Pharmacological concentration of DOX can inhibit the activity and the expression of matrix metalloproteinase 2. The inhibitory effect to proliferation and metastasis of A549 cells may be relation with its inhibitory effect to MMP2.4. Pharmacological concentration of DOX can inhibit the expression of vascular endothelial growth factor(VEGF) and c-jun. The inhibitory effect to MMP2 of DOX may be relation with its inhibitory effect to VEGF and c-jun. AP1 pathway may be a route for DOX to inhibit MMP2 and VEGF of A549.So, we concluded that DOX can inhibit the growth and metastatic protential of human lung carcinoma A549 cells by inhibiting the activity and the expression of matrix metalloproteinase 2. DOX can be taken as an addjactive drug for the therapy of lung cancer.