| The incidence of lung cancer is highest among tumors. Lung cancer is the most dangerous cancer all over the world, which mortality tops the list of all kinds of tumor. The incidence continues to rise at a rate of approximately0.5%per year. Only20%-30%patients with lung cancer can be diagnosed at early stage. Despite recent progress in the diagnosis and the multimodality treatments,prognosis for lung cancer,still,remains unsatisfactory with the overall five-year survival rate only at8to15percent due to metastasis and multidrug resistance.And china has become a country that has the largest number of lung cancer patient.According to histological type,lung cancer can be divided two types:small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).SCLC accounis for more than16%of lung caneer. Small-cell lung cancer is the more aggressive of the two, meaning it can spread quickly to other parts of the body early in the disease. Small-cell lung cancer is notorious for the presence of bony metastases.At present,the main therapy of lung cancer is surgery,combined with chemotherapy and radiotherapy. Chemotherapy is the major mean to treat the advanced cancer, the shortcoming of chemotherapy is to produce great damage not only to the tumor cells, but also the normal cells, especially to blood cells, bone marrow cells, and so on. Its serious side effects impact the quality of life of patients greatly.However, most patients discovered at late stage lost the opportunity to receive operating.Serious tolerance of cancer cells to chemotherapy or radiotherapy resulted in poor prognosis in many cases. It is therefore, urgent to improve the therapeutic effect, to research and to discover a new effective drug in lung cancer therapy.Doxycycline, the important member of the tetracycline family,(2-(amino-hydroxyl-methylidene)-4-dimethylamino-5,10,11,12a-tetrahydroxy-6- methyl-4a,5,5a,6-tetrahydro-4H-tetracene1,3,12-trione) its formula is C22H24N2O8and molecular weight is444.435. Previous reports have demonstrated that Doxycycline had a wide range of pharmacological actions,including anti-inflammatory, anti-microbico、neural protection。 furthermore,the anti-tumor effect of Doxycycline has been reported in a few papers, for example:tongue cancer、prostate cancer、colorectal cancer and so on.In the present study we evaluated the effects of Doxycycline on the Growth of human small cell lung cancer NCI-H446and investigated its mechanism involved in the apoptosis、 proliferation、invasion and metastasis by Doxycycline. It will supply experimental basis for the futher investigation of anti-tumor mechanism of Doxycycline and will provide the theoretical basis for clinical treatment of lung cancer.Methods(1) To investigate the effect of Doxycycline on inhibiting H446cells growth and its mechanism.The lung cancer H446cell culture system was established and was affected by Doxycycline after a certain amount of time. The anti-proliferative activity of Doxycycline in H446cells was detected by CCK8assay and the morphological changes of cells were observed by optical microscope. A total of5concentration gradient were2.5μg/ml、5μ g/ml、10μ g/ml,20μg/ml,40μ g/ml and a total of4time gradient weren24,48,72,96hours, using25μg/ml5-fluorouracil as a positive control drug, and a blank control group (no adding). Relative survival rate was detected, calculation of growth inhibitory rate. Colony formation assay was used to get the cloning formation rate.(2) to investigate doxycycline on the role of human small cell lung cancer H446cells apoptosis.(cells change was obeserved by fluorescence microscopy after doxycycline on H446cells after Hoechst33258staining,(2) Tunel method to detect apoptosis and apoptosis index (3) after AnnexinV-FITC/PI staining flow cytometry for the drug (0,5μg/ml,10μg/ml,20μg/ml Doxycycline and fluorouracil) H446cells of early apoptosis rate were detected.(3) To discuss possible mechanisms of doxycycline induce human small cell lung cancer H446cells apoptosis. With different concentrations of doxycycline on H446cell,(1) After drug technology H446cell apoptosis, using Western blot detect related gene Bax, Bcl-2, caspase3, survivin protein expression level;(2) using Realtime PCR technology to detect intracellular Bax, Bcl-2, caspase3, survivin levels of mRNA.(4)To explore whether doxycycline inhibit human small cell lung cancer H446cells invasion and migration ability.(1) Nick experimental was used to observe doxycycline on H446cell migration ability and (2) Transwell migration distance measurement experimental was used to observe doxycycline on H446cell invasive ability.(5)To investigate Doxycycline on human small cell lung cancer H446cells invasion mechanism,(1) matrix metalloproteinases MMP-2and MMP9and TIMP-2matrix metalloproteinases, and vascular endothelial growth factor VEGF inhibitor levels in the cell supernatant were detected by ELISA.(6)Statistically:The measurement data were expressed as mean±standard deviation(x±s). We used SPSS13.0software for data analysis. The diversity of the design of random data of single factor analysis of variance (One-way ANOVA) multiple comparison method, choose the LSD method; Variance not neat choose when approximate F test Welch method, multiple comparison method select Dunnett’s T3method. P<0.05was considered differences in statistics.Results(1) Different concentrations of doxycycline (2.5μg/ml、μg/ml、10μg/ml、20μg/ml、40μg/ml) after H446cells respectively, the results showed with the increase of doxycycline concentration and action time of H446cell survival rate decreased (p<0.05).24h,48h,72h,96h of IC50respectively (24.10μg/ml10.98μg/ml、8.20μg/ml、8.03μg/ml) of different concentrations of doxycycline, the cell clone formation rate is significantly lower than the control group. Drug concentration is higher, the lower the clone formation rate were(23.43±0.33)%, (11.74±0.64)%,(6.14±0.06)%. were statistically significant (P=0.000, P=0.000, P=0.000) of doxycycline on small cell lung cancer H446has inhibited proliferation effect, and with time and dose dependence. Doxycycline, each dose group compared with the control group had statistical significance.(2)Using Hoechst33258dye, fluorescent microscope observation of cell morphology, flow cytometry Detecting drug under the action of cells apoptosis rate changes. The results showed that the observed under fluorescence microscope, different concentrations of doxycycline treatment after48h, negative control group H446cell nucleus boundaries clear, round or oval, homogeneously blue fluorescence, dyeing shallow; In doxycycline (5,10and20u g/ml) set of visible uneven dyeing of a nucleus, chromatin and appear smaller nuclei procedures, lights is bright blue, nucleus pycnosis, fracture, condensed chromatin distributed granular clumps. In tunel experiments, with0μg/ml、5μg/ml、10μg/ml、20μg/ml doxycycline effect after48h, negative control group have obviously rare nucleus was dyed brown, doxycycline in different concentrations groups compared with negative control group were hyperchromatic visible positive cells, low, medium and high dose group the apoptosis index were(33.17±0.45)%,(51.14±0.96)%、(64.13±0.09)%,compared with negative control group all had statistical significance (P=0.000, P=0.000, P=0.000).0μg/ml、50μg/ml、100μg/ml、200μg/ml doxycycline effect after24h, human small cell lung cancer H446cells of early apoptosis rate were (1.57%±0.06)%,(1.83%±0.06%),(3.17%±0.06)%'(3.43%±0.06)%; Each functional group were statistically significant compared with the control group (P=0.001, P=0.001, P=0.000). Doxycycline induced apoptosis in a dose-response relationship.(3) This research by Western Blot, Real-time PCR detection Bax, Bel-2, caspase-3, survivin gene expression and protein level of change, in order to investigate doxycycline on human small cell lung cancer H446cells apoptotic mechanism, results showed that doxycycline and human small cell lung cancer H446cells after48hours,50μg/ml、100μg/ml、200μg/ml dose of relative content of the Bcl-2mRNA were:0.89±0.04,0.78±0.02,0.39±0.03; The Bel-2in the positive control drug fluorouracil mRNA relative content was0.35±0.01, compared with the negative blank control group was statistically significant (P=0.000). Doxycycline group10ug/ml,20ug/ml dose group of the Bcl-2mRNA H446cells relative content significantly decreased, compared with negative blank control group was statistically significant (P=0.008, P=0.008),5u g/ml dose group no significant difference (P=0.159). BaxmRNA relative content obviously increased, compared with negative blank control group was statistically significant (P=0.001, P=0.001, P=0.047), and the concentration dependence trend, its relative content Bax mRNA respectively is:1.46±0.01,1.54±0.03,1.69±0.06; The relative content of positive control drug fluorouracil in Bax mRNA was1.56±0.04, compared with the negative blank control group was statistically significant (P=0.010). The relative content of Caspase-3mRNA increased significantly, compared with negative blank control group was statistically significant (P=0.001, P=0.001, P=0.019), and a concentration dependent on trend, its relative content of Caspase-3mRNA were:1.44±0.02,1.77±0.04,1.97±0.12; Positive control drug fluorouracil Caspase-3mRNA in relative content was1.93±0.01, compared with the negative blank control group was statistically significant (P=0.036). Survivin mRNA relative content significantly decreased, compared with negative blank control group was statistically significant (P=0.000, P=0.000, P=0.000), and there is concentration dependent on trend, its survivin mRNA relative content are:0.44±0.01,0.31±0.01,0.12±0.00; Positive control drug fluorouracil in the supernatant fluid survivin mRNA relative content was0.93±0.02, compared with the negative blank control group was statistically significant (P=0.000).Western Blot results showed that doxycycline group of low, medium and high dose group of H446cells the Bcl-2protein content significantly decreased, compared with negative blank control group was statistically significant (P=0.001, P=0.001, P=0.014), and the concentration dependence trend, its the Bel-2protein content are:0.75±0.00,0.65±0.03,0.61±0.03; Positive control drug fluorouracil Bel-2protein content was0.62±0.02, compared with the negative blank control group was statistically significant (P=0.003). Bax protein content increased significantly, compared with negative blank control group was statistically significant (P=0.000, P=0.000, P=0.007), and there is concentration dependent on trend, its) Bax protein content are:1.19±0.01,1.55±0.07,2.60±0.14; Positive control drug fluorouracil) Bax protein content was2.11±0.06, compared with the negative blank control group was statistically significant (P=0.003). Caspase3protein content increased significantly, compared with negative blank control group was statistically significant (P=0.000, P=0.000, P=0.004), and there is concentration dependent on trend, its the protein content of caspase3are:1.35±0.00,1.77±0.03mm,1.86±0.04mm; Positive control drug fluorouracil caspase protein content was1.10±0.03-3, compared with the negative blank control group was statistically significant (P=0.001). Survivin protein content significantly decreased, compared with negative blank control group was statistically significant (P=0.000, P=0.000, P=0.003), and the concentration dependence trend, its survivin protein content, respectively is:0.84±0.01,0.64±0.08mm,0.24±0.06mm; Positive control drug fluorouracil survivin protein content was0.35±0.04, compared with the negative blank control group was statistically significant (P=0.001). Were known as the inhibition of H446cells in the role of survivin protein content.(4) Invasion and metastasis is the most essential feature of malignant tumor. This research adopts experiment and transwell cell scratch test, observation of drug on H446cell invasive and migration ability, the influence of the results showed that doxycycline on human small cell lung cancer H446cells to inhibit the action of the attack. With5μg/ml、10μg/ml、20μg/ml doxycycline effect after48h, H446cells migration distance by0.56±0.00in the control group were reduced to0.43±0.01,0.32±0.01,0.11±0.00, drug groups all have significant difference compared with the control group statistically significant (P=0.000, P=0.000, P=0.000); Fluorouracil and positive control group results are statistically significant (P=0.000); Respectively to5μg/ml、10μg/ml、20μg/ml doxycycline effect after48h, through the artificial basement membrane H446cells reduced by471.33±2.08in the control group, respectively, and265.33±4.04,194.67±3.51and3.51±3.61; Between groups with the negative control group had significantly statistical difference (P<0.01).(5) Impact study doxycycline H446cell invasive and migration ability of the possible mechanism. Using ELISA method to detect cell supernatant fluid of matrix metalloproteinases MMP-2and MMP-9, matrix metalloproteinases inhibitor TIMP-2and vascular endothelial growth factor VEGF levels. Results showed that doxycycline group of low, medium and high dose group of H446cells supernatant in the expression of MMP-2significantly decreased, compared with negative blank control group was statistically significant (P=0.000, P=0.000, P=0.000), and the concentration dependence trend, its amount of the expression of MMP-2are:19.19±0.23ng/ml,16.41±0.14ng/ml,14.71±0.32ng/ml; Positive control drug fluorouracil the expression of MMP-2in the supernatant fluid amount is15.35±0.51ng/ml, compared with the negative blank control group was statistically significant (P=0.000). The expression of MMP-9is decreased obviously compared with negative blank control group was statistically significant (P=0.022, P=0.022, P=0.000), and there is concentration dependent on trend, its amount of the expression of MMP-9are:2.81±0.16ng/ml,2.05±0.14ng/ml,1.64±0.04ng/ml; Positive control drug fluorouracil supernatant fluid of the expression of MMP-9was1.42±0.05ng/ml, compared with the negative blank control group was statistically significant (P=0.000). Expression of TIMP-2significantly increased, compared with negative blank control group was statistically significant (P=0.005, P=0.005, P=0.000), and the concentration dependence trend, its amount of the expression of TIMP-2are:0.69±0.06ng/ml,0.99±0.07ng/ml,1.37±0.02ng/ml; Positive control drug fluorouracil expression of TIMP-2in the supernatant fluid amount is1.21±0.07ng/ml, compared with the negative blank control group was statistically significant (P=0.000). The expression of VEGF decreased significantly, compared with negative blank control group was statistically significant (P=0.000, P=0.000, P=0.000), and there is concentration dependent on trend, the expression of VEGF are:286.88±3.14ng/ml,260.37±3.66ng/ml,230.93±5.30ng/ml; Positive control drug fluorouracil the expression of VEGF in the supernatant fluid amount is218.77±10.30ng/ml, compared with the negative blank control group was statistically significant (P= 0.000).ConclusionDoxycycline for small cell lung cancer H446cells has antiproliferative effect; Doxycycline in5u g/ml-20u g/ml concentration range on small cell lung cancer H446cells have different degree of inducing apoptosis, with a dose-response relationship; Possible mechanisms of apoptosis induced by doxycycline is related with adjustment apoptosis gene:Bax, Bel-2, caspase3, survivin; Doxycycline can inhibit human small cell lung cancer H446cell migration and invasion; Doxycycline can adjust levels of MMP-2and MMP-9, tissue matrix metalloproteinases inhibitor TIMP-2and expression of vascular endothelial growth factor VEGF in human small cell lung cancer H446cells to inhibit invasio n and migration. In conclusion, Doxycycline plays a role in induction of the apoptosis and inhibits the proliferation, invasion, metastasis of human small cell lung cancer H446cells, has a potential development prospect as a new drug for lung cancer treatment. |
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