多西环素对人小细胞肺癌H446细胞的抗肿瘤作用及其机制研究
摘要
背景和目的
肺癌是临床发病率最高的恶性肿瘤,其死亡率居于各种恶性肿瘤之首,且全世界肺癌发病率还在以每年0.5%的速度增长。仅有20%-30%的肺癌患者可以早期得以诊断。尽管肺癌的诊断和治疗手段不断取得新的进展,但由于肿瘤转移及多药耐药等问题,肺癌患者预后仍不佳,五年生存率仅为8-15%。中国也己成为世界上第一肺癌大国。按组织学类型可将肺癌分为两大类：小细胞肺癌和非小细胞肺癌。小细胞肺癌占肺癌总数的16%,且更具侵袭性,在早期就能扩散到其它器官,且往往伴有骨转移。
目前肺癌的综合治疗措施仍是以手术为主,化疗、放疗为辅。化疗是目前治疗中、晚期肿瘤的主要手段,缺点是对瘤体细胞产生杀伤作用的同时,对正常组织细胞也同样产生杀伤作用,特别是对生长迅速的血细胞、骨髓细胞等损害更大。化疗严重的副作用对患者的生活质量有很大的影响。由于肺癌发现时多为晚期,许多病人已丧失了手术机会,同时化、放疗耐受现象又较严重,预后很差。因此寻求新的有效的肺癌治疗药物,进一步提高临床疗效,己成为厄待解决的临床难题之一。
多西环素是四环素类抗生素,其分子式为C22H24N2O8,分子量为444.435。具有抗炎、抗菌、神经保护等广泛的药理作用。新近研究报道其对于多种肿瘤具有一定的细胞毒作用,包括舌癌、前列腺癌、直肠癌等。目前多西环素对小细胞肺癌细胞是否有抑制作用的研究尚未见报道。本实验通过研究多西环素对小细胞肺腺癌细胞系NCI-H446增殖、凋亡、侵袭和转移的影响,并探讨其潜在的作用机制,以期为深入阐明多西环素的抗肿瘤机制提供实验基础,为多西环素应用于临床治疗肺癌提供理论依据。
方法
(1)探讨多西环素对体外人小细胞肺癌H446细胞增殖的影响及其机制。①建立人小细胞肺癌H446细胞培养体系；多西环素作用于小细胞肺癌H446细胞一定时间段后,光镜下观察细胞形态学变化；②应用CCK-8法对各组细胞增殖抑制率进行检测,共设2.5μg/ml、5μg/ml、10μg/ml、20μg/ml、40μg/ml5个浓度梯度,并设24、48、72、96小时共4个时间梯度；选用25μg/ml的5-氟尿嘧啶作为阳性对照药物,同时设置空白对照组(不加药),检测各组相对生存率,计算增殖抑制率③平板克隆形成实验检测药物处理后各组的克隆形成率；
(2)探讨多西环素对人小细胞肺癌H446细胞调亡的影响。①多西环素作用于H446细胞后,以Hoechst33258染色后,荧光显微镜下观察细胞微观结构的变化。②Tunel染色法检测细胞凋亡并计算凋亡指数③AnnexinV-FITC/PI染色流式细胞术对药物(0.5μg/ml、10μg/ml、20μg/ml的多西环素及阳性对照药物氟尿嘧啶)作用后H446细胞的早期调亡率进行检测。
(3)探讨多西环素诱导人小细胞肺癌H446细胞凋亡的可能机制。以不同浓度的多西环素作用于H446细胞后,①采用Western blot法检测药物作用后H446细胞内凋亡相关蛋白Bax、Bcl-2、caspase3、survivin表达水平的变化；②采用Real-time PCR技术检测多西环素药物刺激对细胞内Bax、Bcl-2、caspase3、 survivin的mRNA表达水平的影响。
(4)探讨多西环素是否具有抑制人小细胞肺癌H446细胞迁移和侵袭的能力。①采用细胞划痕实验通过测量迁移距离评价多西环素对H446细胞的迁移能力的影响②采用Transwell实验观察多西环素对H446细胞的侵袭能力的影响。
(5)探讨多西环素影响人小细胞肺癌H446细胞侵袭作用的机制：①采用ELISA法检测各组细胞上清液中基质金属蛋白酶MMP-2、MMP-9及组织基质金属蛋白酶抑制剂TIMP-2的浓度水平②采用ELISA法检测各组细胞上清液中血管内皮生长因子VEGF的水平。
(6)统计方法：结果数据以均数±标准差(x±s)表示。采用SPSS13.0软件进行数据分析。随机设计的多样本资料进行单因素方差分析(One-way ANOVA)多重比较时,方差齐性时,选择LSD法,方差不齐时选择近似F检验Welch法,多重比较方法选择Dunnett's T3法。P<0.05时认为有统计(学)差异。
结果
(1)不同浓度的多西环素(2.5μg/ml、5μg/ml、10μg/ml、20μg/ml、40μg/ml)分别作用H446细胞不同时间段后,结果表明随着多西环素浓度和作用时间的增加,H446细胞存活率降低(p<0.05)。多西环素作用24h、48h、72h、96h的IC50分别为24.10μg/ml、10.98μg/ml、8.20μg/ml、8.03μg/ml。不同浓度的多西环素作用后,加药组的克隆形成率明显低于对照组。药物浓度越高,克隆形成率越低。5μg/ml、10μg/ml、20μg/ml组的克隆形成率分别为(23.43±0.33)%、(11.74±0.64)%及(6.14±0.06)%,差异具有统计学意义(P=0.000,P=0.000,P=0.000)。上述结果表明多西环素可时间-浓度依赖性的抑制人小细胞肺癌H446细胞的增殖。
(2)利用Hoechst33258染色,荧光显微镜下观察细胞的形态；流式细胞术检测药物作用后细胞的凋亡率变化。结果：荧光显微镜下可见,阴性对照组H446细胞核界限清晰,圆形或椭圆形,呈均匀蓝色荧光,染色较浅；不同浓度的多西环素(5、10、20μg/ml)处理48h后,可见细胞核染色不均一,胞核固缩、碎裂,染色质凝集呈颗粒团状分布；在tunel实验中,阴性对照组少见明显的细胞核被染成棕褐色。5μg/ml、10μg/ml、20μg/ml的多西环素作用48h后,与阴性对照组相比均可见被深染的阳性细胞,低、中、高剂量组的凋亡指数分别为(33.17±0.45)%、(51.14±0.96)%、(64.13±0.09)%,与阴性对照组比较均具有统计学意义(P=0.000, P=0.OOO,P=0.000);0μg/ml、5μg/ml、1Oμg/ml、20μg/ml的多西环素作用24h后,人小细胞肺癌H446细胞的早期凋亡率分别为(1.57%±0.06)%、(1.83%±0.06)%、(3.17%±0.06)%和(3.43%±0.06)%。各加药组与空白组相比均有统计学差异(P=0.001,P=0.000,P=0.000)。上述形态学变化及凋亡率结果均表明多西环素可浓度依赖性的诱导人小细胞肺癌H446细胞的凋亡。
(3)通过Western Blot、Real-time PCR法检测Bax、Bcl-2、caspase-3、survivin的蛋白和：mRNA表达水平的变化,以探讨多西环素诱导人小细胞肺癌H446细胞凋亡作用的可能的机制。结果：多西环素作用于人小细胞肺癌H446细胞48小时后,10μg/ml、20μg/ml多西环素组H446细胞中Bcl-2mRNA的相对含量明显降低,相对含量分别为：0.78±0.02及0.39±0.03,与阴性空白对照组比较有统计学意义(P=0.008, P=0.003)。5μg/ml剂量组mRNA的相对含量为0.89±0.04,与阴性空白对照组比较无显著性差异(P=0.159)。阳性对照药物氟尿嘧啶中Bcl-2mRNA的相对含量为0.35±0.01,与阴性空白对照组相比有统计学意义(P=0.000); Bax的mRNA的相对含量明显升高,5μg/ml、10μg/ml、20μg/ml多西环素组Bax的mRNA的相对含量分别为：1.46±0.01、1.54±0.03、1.69±0.06,与阴性空白对照组比较有统计学意义(P=0.001,P=0.007,P=0.047)。阳性对照药物氟尿嘧啶组Bax的]mRNA的相对含量为1.56±0.04,与阴性空白对照组相比有统计学意义(P=0.010); Caspase-3mRNA的相对含量明显升高,且有浓度依赖趋势。5μg/ml、10μg/ml、20μg/ml多西环素组Caspase-3mRNA的相对含量分别为：1.44±0.02、1.77±0.04、1.97±0.12,与阴性空白对照组比有统计学意义(P=0.001,P=0.035,P=0.019)。阳性对照药物氟尿嘧啶中Caspase-3mRNA的相对含量为1.93±0.01,与阴性空白对照组相比有统计学意义(P=0.036); survivin mRNA的相对含量明显降低,且有浓度依赖趋势。5μg/ml、10μg/ml、20μg/ml多西环素组survivin mRNA的相对含量分别为：0.444±0.01、0.31±0.01、0.12±0.00,与阴性空白对照组比较有统计学意义(P=0.000,P=0.000,P=0.000)。阳性对照药物氟尿嘧啶组survivin mRNA的相对含量为0.93±0.02,与阴性空白对照组相比有统计学意义(P=0.000)。
Western Blot结果显示：多西环素组组H446细胞中Bcl-2的蛋白表达量明显降低,且有浓度依赖趋势。低、中、高剂量Bcl-2的蛋白相对含量分别为：0.75±0.00、0.65±0.03、0.61±0.03,与阴性空白对照组比较有统计学意义(P=0.001,P=0.013,P=0.014)。阳性对照药物氟尿嘧啶组Bcl-2的蛋白含量为0.62±0.02,与阴性空白对照组相比有统计学意义(P=0.003)；多西环素组组H446细胞中Bax的蛋白含量明显增高,且有浓度依赖趋势。低、中、高剂量组Bax的蛋白相对含量分别为：1.19±0.01、1.55±0.07、2.60±0.14，与阴性空白对照组比较有统计学意义(P=0.000，P=0.009,P=0.007)。阳性对照药物氟尿嘧啶组Bax的蛋白相对含量为2.11±0.06，与阴性空白对照组相比有统计学意义(P=0.003)；多西环素组组H446细胞中caspaSe-3的蛋白含量明显增高,且有浓度依赖趋势。低、中、高剂量组caspase-3的蛋白相对含量分别为1.35±0.00、1.77±0.03、1.86±0.04，与阴性空白对照组比较有统计学意义(P=0.000,P=0.003,P=0.004)。阳性对照药物氟尿嘧啶组caspase-3的蛋白相对含量为1.10-±0.03，与阴性空白对照组相比有统计学意义(P=0.001)；多西环素组组H446细胞中survivin的蛋白含量明显降低,且有浓度依赖趋势。低、中、高剂量组survivin的蛋白相对含量分别为0.84±0.01、0.64±0.08、0.24±0.06，与阴性空白对照组比较有统计学意义(P=0.000,P=0.017、P=0.003)。阳性对照药物氟尿嘧啶survivin的蛋白含量为0.35±0.04,与阴性空白对照组相比有统计学意义(P=0.001)。
(4)侵袭、转移是恶性肿瘤最本质的特性。本研究采用细胞划痕实验及stanswell实验,观察多西环素对H446细胞侵袭性和迁移能力的影响,结果表明,多西环素具有抑制人小细胞肺癌H446细胞侵袭能力的作用。5μ/ml、10μg/ml、20μg/ml多西环素作用48h后,H446细胞的迁移距离由对照组的0.56±0.00分别缩短至0.43+0.01、0.32±0.01、0.11±0.00，与对照组结果比较差异均具显著统计学意义(P=0.000,P=0.000,P=0.000)。阳性对照组(氟尿嘧啶)与对照组结果比较结果也具有统计学意义(P=0.000)；5μ/ml、10μg/ml、20μg/ml多西环素作用48h后,穿过人工基底膜的H446细胞数由对照组的471.33±2.08分别减少至265.33±4.04、194.67±3.51和88.00±3.61；各组与阴性对照组比均有显著统计学差异(P均<0.01)。
(5)研究多西环素影响H446细胞侵袭性和迁移能力的可能的机制。采用ELISA法检测细胞上清液中基质金属蛋白酶MMP-2、MMP-9、组织基质金属蛋白酶抑制剂TIMP-2及水平血管内皮生长因子VEGF的浓度水平。结果表明：多西环素组H446细胞上清液中MMP-2的含量明显降低,且有浓度依赖趋势。低、中、高剂量组MMP-2的含量分别为19.19±0.23ng/ml、16.41±0.14ng/ml、14.71±0.32ng/ml,与阴性空白对照组比较有统计学意义(P=0.000,P=0.000,P=0.000)。阳性对照药物氟尿嘧啶组上清液中MMP-2的含量为15.35±0.51ng/ml,与阴性空白对照组相比有统计学意义(P=0.000)；多西环素组H446细胞上清液中MMP-9的含量明显降低,且有浓度依赖趋势。低、中、高剂量组MMP-9的含量分别为2.81±0.16ng/ml、2.05±0.14ng/ml、1.64±0.04ng/ml,与阴性空白对照组比较有统计学意义(P=0.022,P=0.004,P=0.000)。阳性对照药物氟尿嘧啶组上清液中MMP-9的含量为1.42±0.05ng/ml,与阴性空白对照组相比有统计学意义(P=0.000)；多西环素组H446细胞上清液中TIMP-2的含量明显增加,,且有浓度依赖趋势。低、中、高剂量组TIMP-2的含量分别为0.69±0.06ng/ml、0.99±0.07ng/ml、1.37±0.02ng/ml,与阴性空白对照组比较有统计学意义(P=0.005,P=0.000,P=0.000)。阳性对照药物氟尿嘧啶上清液中TIMP-2的含量为1.21±0.07ng/ml,与阴性空白对照组相比有统计学意义(P=0.000)；多西环素组H446细胞上清液中VEGF的含量明显减少,且有浓度依赖趋势。低、中、高剂量组VEGF的含量分别为286.88±3.14ng/ml、260.37±3.66ng/ml、230.93±5.30ng/ml,与阴性空白对照组比较有统计学意义(P=0.000,P=0.000,P=0.000)。阳性对照药物氟尿嘧啶上清液中VEGF的含量为218.77±10.30ng/ml,与阴性空白对照组相比有统计学意义(P=0.000)。
结论
多西环素对小细胞肺癌H446细胞的增殖具有抑制作用；5μg/ml-20μg/ml浓度范围内多西环素可浓度依赖性的诱导人小细胞肺癌H446细胞调亡；多西环素诱导细胞凋亡的作用可能与调节凋亡基因Bax、Bcl-2、caspase3、survivin有关；多西环素具有抑制人小细胞肺癌H446细胞迁移与侵袭的能力；多西环素可通过调节人小细胞肺癌H446细胞中MMP-2、MMP-9、组织基质金属蛋白酶抑制剂TIMP-2及水平血管内皮生长因子VEGF的表达进而抑制侵袭和迁移。综上,多西环素对人小细胞肺癌H446细胞具有诱导凋亡与抑制增殖、侵袭、转移的作用,具有潜在的作为一种肺癌治疗新药的研发前景。
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)%和(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).
Conclusion
Doxycycline 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.
肺癌是临床发病率最高的恶性肿瘤,其死亡率居于各种恶性肿瘤之首,且全世界肺癌发病率还在以每年0.5%的速度增长。仅有20%-30%的肺癌患者可以早期得以诊断。尽管肺癌的诊断和治疗手段不断取得新的进展,但由于肿瘤转移及多药耐药等问题,肺癌患者预后仍不佳,五年生存率仅为8-15%。中国也己成为世界上第一肺癌大国。按组织学类型可将肺癌分为两大类：小细胞肺癌和非小细胞肺癌。小细胞肺癌占肺癌总数的16%,且更具侵袭性,在早期就能扩散到其它器官,且往往伴有骨转移。
目前肺癌的综合治疗措施仍是以手术为主,化疗、放疗为辅。化疗是目前治疗中、晚期肿瘤的主要手段,缺点是对瘤体细胞产生杀伤作用的同时,对正常组织细胞也同样产生杀伤作用,特别是对生长迅速的血细胞、骨髓细胞等损害更大。化疗严重的副作用对患者的生活质量有很大的影响。由于肺癌发现时多为晚期,许多病人已丧失了手术机会,同时化、放疗耐受现象又较严重,预后很差。因此寻求新的有效的肺癌治疗药物,进一步提高临床疗效,己成为厄待解决的临床难题之一。
多西环素是四环素类抗生素,其分子式为C22H24N2O8,分子量为444.435。具有抗炎、抗菌、神经保护等广泛的药理作用。新近研究报道其对于多种肿瘤具有一定的细胞毒作用,包括舌癌、前列腺癌、直肠癌等。目前多西环素对小细胞肺癌细胞是否有抑制作用的研究尚未见报道。本实验通过研究多西环素对小细胞肺腺癌细胞系NCI-H446增殖、凋亡、侵袭和转移的影响,并探讨其潜在的作用机制,以期为深入阐明多西环素的抗肿瘤机制提供实验基础,为多西环素应用于临床治疗肺癌提供理论依据。
方法
(1)探讨多西环素对体外人小细胞肺癌H446细胞增殖的影响及其机制。①建立人小细胞肺癌H446细胞培养体系；多西环素作用于小细胞肺癌H446细胞一定时间段后,光镜下观察细胞形态学变化；②应用CCK-8法对各组细胞增殖抑制率进行检测,共设2.5μg/ml、5μg/ml、10μg/ml、20μg/ml、40μg/ml5个浓度梯度,并设24、48、72、96小时共4个时间梯度；选用25μg/ml的5-氟尿嘧啶作为阳性对照药物,同时设置空白对照组(不加药),检测各组相对生存率,计算增殖抑制率③平板克隆形成实验检测药物处理后各组的克隆形成率；
(2)探讨多西环素对人小细胞肺癌H446细胞调亡的影响。①多西环素作用于H446细胞后,以Hoechst33258染色后,荧光显微镜下观察细胞微观结构的变化。②Tunel染色法检测细胞凋亡并计算凋亡指数③AnnexinV-FITC/PI染色流式细胞术对药物(0.5μg/ml、10μg/ml、20μg/ml的多西环素及阳性对照药物氟尿嘧啶)作用后H446细胞的早期调亡率进行检测。
(3)探讨多西环素诱导人小细胞肺癌H446细胞凋亡的可能机制。以不同浓度的多西环素作用于H446细胞后,①采用Western blot法检测药物作用后H446细胞内凋亡相关蛋白Bax、Bcl-2、caspase3、survivin表达水平的变化；②采用Real-time PCR技术检测多西环素药物刺激对细胞内Bax、Bcl-2、caspase3、 survivin的mRNA表达水平的影响。
(4)探讨多西环素是否具有抑制人小细胞肺癌H446细胞迁移和侵袭的能力。①采用细胞划痕实验通过测量迁移距离评价多西环素对H446细胞的迁移能力的影响②采用Transwell实验观察多西环素对H446细胞的侵袭能力的影响。
(5)探讨多西环素影响人小细胞肺癌H446细胞侵袭作用的机制：①采用ELISA法检测各组细胞上清液中基质金属蛋白酶MMP-2、MMP-9及组织基质金属蛋白酶抑制剂TIMP-2的浓度水平②采用ELISA法检测各组细胞上清液中血管内皮生长因子VEGF的水平。
(6)统计方法：结果数据以均数±标准差(x±s)表示。采用SPSS13.0软件进行数据分析。随机设计的多样本资料进行单因素方差分析(One-way ANOVA)多重比较时,方差齐性时,选择LSD法,方差不齐时选择近似F检验Welch法,多重比较方法选择Dunnett's T3法。P<0.05时认为有统计(学)差异。
结果
(1)不同浓度的多西环素(2.5μg/ml、5μg/ml、10μg/ml、20μg/ml、40μg/ml)分别作用H446细胞不同时间段后,结果表明随着多西环素浓度和作用时间的增加,H446细胞存活率降低(p<0.05)。多西环素作用24h、48h、72h、96h的IC50分别为24.10μg/ml、10.98μg/ml、8.20μg/ml、8.03μg/ml。不同浓度的多西环素作用后,加药组的克隆形成率明显低于对照组。药物浓度越高,克隆形成率越低。5μg/ml、10μg/ml、20μg/ml组的克隆形成率分别为(23.43±0.33)%、(11.74±0.64)%及(6.14±0.06)%,差异具有统计学意义(P=0.000,P=0.000,P=0.000)。上述结果表明多西环素可时间-浓度依赖性的抑制人小细胞肺癌H446细胞的增殖。
(2)利用Hoechst33258染色,荧光显微镜下观察细胞的形态；流式细胞术检测药物作用后细胞的凋亡率变化。结果：荧光显微镜下可见,阴性对照组H446细胞核界限清晰,圆形或椭圆形,呈均匀蓝色荧光,染色较浅；不同浓度的多西环素(5、10、20μg/ml)处理48h后,可见细胞核染色不均一,胞核固缩、碎裂,染色质凝集呈颗粒团状分布；在tunel实验中,阴性对照组少见明显的细胞核被染成棕褐色。5μg/ml、10μg/ml、20μg/ml的多西环素作用48h后,与阴性对照组相比均可见被深染的阳性细胞,低、中、高剂量组的凋亡指数分别为(33.17±0.45)%、(51.14±0.96)%、(64.13±0.09)%,与阴性对照组比较均具有统计学意义(P=0.000, P=0.OOO,P=0.000);0μg/ml、5μg/ml、1Oμg/ml、20μg/ml的多西环素作用24h后,人小细胞肺癌H446细胞的早期凋亡率分别为(1.57%±0.06)%、(1.83%±0.06)%、(3.17%±0.06)%和(3.43%±0.06)%。各加药组与空白组相比均有统计学差异(P=0.001,P=0.000,P=0.000)。上述形态学变化及凋亡率结果均表明多西环素可浓度依赖性的诱导人小细胞肺癌H446细胞的凋亡。
(3)通过Western Blot、Real-time PCR法检测Bax、Bcl-2、caspase-3、survivin的蛋白和：mRNA表达水平的变化,以探讨多西环素诱导人小细胞肺癌H446细胞凋亡作用的可能的机制。结果：多西环素作用于人小细胞肺癌H446细胞48小时后,10μg/ml、20μg/ml多西环素组H446细胞中Bcl-2mRNA的相对含量明显降低,相对含量分别为：0.78±0.02及0.39±0.03,与阴性空白对照组比较有统计学意义(P=0.008, P=0.003)。5μg/ml剂量组mRNA的相对含量为0.89±0.04,与阴性空白对照组比较无显著性差异(P=0.159)。阳性对照药物氟尿嘧啶中Bcl-2mRNA的相对含量为0.35±0.01,与阴性空白对照组相比有统计学意义(P=0.000); Bax的mRNA的相对含量明显升高,5μg/ml、10μg/ml、20μg/ml多西环素组Bax的mRNA的相对含量分别为：1.46±0.01、1.54±0.03、1.69±0.06,与阴性空白对照组比较有统计学意义(P=0.001,P=0.007,P=0.047)。阳性对照药物氟尿嘧啶组Bax的]mRNA的相对含量为1.56±0.04,与阴性空白对照组相比有统计学意义(P=0.010); Caspase-3mRNA的相对含量明显升高,且有浓度依赖趋势。5μg/ml、10μg/ml、20μg/ml多西环素组Caspase-3mRNA的相对含量分别为：1.44±0.02、1.77±0.04、1.97±0.12,与阴性空白对照组比有统计学意义(P=0.001,P=0.035,P=0.019)。阳性对照药物氟尿嘧啶中Caspase-3mRNA的相对含量为1.93±0.01,与阴性空白对照组相比有统计学意义(P=0.036); survivin mRNA的相对含量明显降低,且有浓度依赖趋势。5μg/ml、10μg/ml、20μg/ml多西环素组survivin mRNA的相对含量分别为：0.444±0.01、0.31±0.01、0.12±0.00,与阴性空白对照组比较有统计学意义(P=0.000,P=0.000,P=0.000)。阳性对照药物氟尿嘧啶组survivin mRNA的相对含量为0.93±0.02,与阴性空白对照组相比有统计学意义(P=0.000)。
Western Blot结果显示：多西环素组组H446细胞中Bcl-2的蛋白表达量明显降低,且有浓度依赖趋势。低、中、高剂量Bcl-2的蛋白相对含量分别为：0.75±0.00、0.65±0.03、0.61±0.03,与阴性空白对照组比较有统计学意义(P=0.001,P=0.013,P=0.014)。阳性对照药物氟尿嘧啶组Bcl-2的蛋白含量为0.62±0.02,与阴性空白对照组相比有统计学意义(P=0.003)；多西环素组组H446细胞中Bax的蛋白含量明显增高,且有浓度依赖趋势。低、中、高剂量组Bax的蛋白相对含量分别为：1.19±0.01、1.55±0.07、2.60±0.14，与阴性空白对照组比较有统计学意义(P=0.000，P=0.009,P=0.007)。阳性对照药物氟尿嘧啶组Bax的蛋白相对含量为2.11±0.06，与阴性空白对照组相比有统计学意义(P=0.003)；多西环素组组H446细胞中caspaSe-3的蛋白含量明显增高,且有浓度依赖趋势。低、中、高剂量组caspase-3的蛋白相对含量分别为1.35±0.00、1.77±0.03、1.86±0.04，与阴性空白对照组比较有统计学意义(P=0.000,P=0.003,P=0.004)。阳性对照药物氟尿嘧啶组caspase-3的蛋白相对含量为1.10-±0.03，与阴性空白对照组相比有统计学意义(P=0.001)；多西环素组组H446细胞中survivin的蛋白含量明显降低,且有浓度依赖趋势。低、中、高剂量组survivin的蛋白相对含量分别为0.84±0.01、0.64±0.08、0.24±0.06，与阴性空白对照组比较有统计学意义(P=0.000,P=0.017、P=0.003)。阳性对照药物氟尿嘧啶survivin的蛋白含量为0.35±0.04,与阴性空白对照组相比有统计学意义(P=0.001)。
(4)侵袭、转移是恶性肿瘤最本质的特性。本研究采用细胞划痕实验及stanswell实验,观察多西环素对H446细胞侵袭性和迁移能力的影响,结果表明,多西环素具有抑制人小细胞肺癌H446细胞侵袭能力的作用。5μ/ml、10μg/ml、20μg/ml多西环素作用48h后,H446细胞的迁移距离由对照组的0.56±0.00分别缩短至0.43+0.01、0.32±0.01、0.11±0.00，与对照组结果比较差异均具显著统计学意义(P=0.000,P=0.000,P=0.000)。阳性对照组(氟尿嘧啶)与对照组结果比较结果也具有统计学意义(P=0.000)；5μ/ml、10μg/ml、20μg/ml多西环素作用48h后,穿过人工基底膜的H446细胞数由对照组的471.33±2.08分别减少至265.33±4.04、194.67±3.51和88.00±3.61；各组与阴性对照组比均有显著统计学差异(P均<0.01)。
(5)研究多西环素影响H446细胞侵袭性和迁移能力的可能的机制。采用ELISA法检测细胞上清液中基质金属蛋白酶MMP-2、MMP-9、组织基质金属蛋白酶抑制剂TIMP-2及水平血管内皮生长因子VEGF的浓度水平。结果表明：多西环素组H446细胞上清液中MMP-2的含量明显降低,且有浓度依赖趋势。低、中、高剂量组MMP-2的含量分别为19.19±0.23ng/ml、16.41±0.14ng/ml、14.71±0.32ng/ml,与阴性空白对照组比较有统计学意义(P=0.000,P=0.000,P=0.000)。阳性对照药物氟尿嘧啶组上清液中MMP-2的含量为15.35±0.51ng/ml,与阴性空白对照组相比有统计学意义(P=0.000)；多西环素组H446细胞上清液中MMP-9的含量明显降低,且有浓度依赖趋势。低、中、高剂量组MMP-9的含量分别为2.81±0.16ng/ml、2.05±0.14ng/ml、1.64±0.04ng/ml,与阴性空白对照组比较有统计学意义(P=0.022,P=0.004,P=0.000)。阳性对照药物氟尿嘧啶组上清液中MMP-9的含量为1.42±0.05ng/ml,与阴性空白对照组相比有统计学意义(P=0.000)；多西环素组H446细胞上清液中TIMP-2的含量明显增加,,且有浓度依赖趋势。低、中、高剂量组TIMP-2的含量分别为0.69±0.06ng/ml、0.99±0.07ng/ml、1.37±0.02ng/ml,与阴性空白对照组比较有统计学意义(P=0.005,P=0.000,P=0.000)。阳性对照药物氟尿嘧啶上清液中TIMP-2的含量为1.21±0.07ng/ml,与阴性空白对照组相比有统计学意义(P=0.000)；多西环素组H446细胞上清液中VEGF的含量明显减少,且有浓度依赖趋势。低、中、高剂量组VEGF的含量分别为286.88±3.14ng/ml、260.37±3.66ng/ml、230.93±5.30ng/ml,与阴性空白对照组比较有统计学意义(P=0.000,P=0.000,P=0.000)。阳性对照药物氟尿嘧啶上清液中VEGF的含量为218.77±10.30ng/ml,与阴性空白对照组相比有统计学意义(P=0.000)。
结论
多西环素对小细胞肺癌H446细胞的增殖具有抑制作用；5μg/ml-20μg/ml浓度范围内多西环素可浓度依赖性的诱导人小细胞肺癌H446细胞调亡；多西环素诱导细胞凋亡的作用可能与调节凋亡基因Bax、Bcl-2、caspase3、survivin有关；多西环素具有抑制人小细胞肺癌H446细胞迁移与侵袭的能力；多西环素可通过调节人小细胞肺癌H446细胞中MMP-2、MMP-9、组织基质金属蛋白酶抑制剂TIMP-2及水平血管内皮生长因子VEGF的表达进而抑制侵袭和迁移。综上,多西环素对人小细胞肺癌H446细胞具有诱导凋亡与抑制增殖、侵袭、转移的作用,具有潜在的作为一种肺癌治疗新药的研发前景。
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)%和(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).
Conclusion
Doxycycline 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.
引文
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