榄香烯对乏氧肺癌细胞的放射增敏作用与mTOR及HIF-1α/Survivin通路的相关性研究
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摘要
肺癌是严重威胁人类生命的一种临床常见的恶性肿瘤，每年约有130万人死于该病，其中以非小细胞肺癌居多，约占80％。由于肺癌患者在确诊时多已经是中晚期，主要还是接受化疗、放疗等综合治疗。研究发现，绝大多数肿瘤组织内存在有不同程度的乏氧细胞，而乏氧细胞对放射线是抗拒的，这也是造成肿瘤放疗失败和复发的根源。为克服肿瘤乏氧状况，人们间接地或直接地采用增加肿瘤内氧含量的方法，治疗效果仍不尽人意。因此，开发与研究肿瘤乏氧细胞放射增敏剂提高放射敏感性已成为肿瘤放疗研究的热点课题。
榄香烯(Elemene)是一种具有抗肿瘤和放射增敏作用的中药，众多基础及临床实验研究也已经证实。课题组前期在研究榄香烯对肺腺癌A549细胞的放射增敏实验中发现，其通过增加肿瘤细胞的G2/M期阻滞、下调Bcl-2和上调p53基因表达促进细胞凋亡而发挥放射增敏作用，其增敏机制也与抑制细胞端粒酶活性、诱导细胞DNA双链和单链损伤及抑制损伤修复有关。体外实验也证实榄香烯联合放疗可以改善肿瘤乏氧状态而发挥放射增敏作用。检索文献发现，对榄香烯放射增敏作用相关的信号通路，研究报道甚少。
哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）、缺氧诱导因子-1α（hypoxia inducible factor-1alpha, HIF-1α）以及生存素（Survivin）在恶性肿瘤组织中有较高表达，与肿瘤细胞的凋亡有着密切联系，而且mTOR和HIF-1α还与恶性肿瘤细胞的增殖、侵袭和转移有关。课题组前期在体内肺癌裸鼠移植瘤实验中发现榄香烯联合放疗可以降低放射诱导的HIF-1α、Survivin表达增多，而且对mTOR的抑制作用比单独榄香烯治疗时强，从而增加移植瘤组织内肺癌细胞的凋亡，提示mTOR、HIF-1α、Survivin可能是榄香烯放射增敏的作用靶点，但是否通过作用于mTOR，进而影响HIF-1α/Survivin的表达，暂时还未明确。以往体外研究榄香烯的放射增敏作用多在常氧条件下进行，因此本实验拟观察榄香烯对乏氧肺腺癌细胞株（A549）和肺鳞癌细胞株（H520）的放射增敏作用，探讨榄香烯联合照射对乏氧肺癌细胞中mTOR、HIF-1α和Survivin表达的影响，以及mTOR和HIF-1α/Survivin通路对肺癌细胞活性的调节作用，和与榄香烯放射增敏作用的关系。
目的：1、选用低浓度榄香烯作用于乏氧A549和H520细胞，观察榄香烯对乏氧肺癌细胞株的放射增敏作用；研究榄香烯联合照射对乏氧A549和H520细胞中mTOR、HIF-1α和Survivin表达的影响。
2、研究mTOR和HIF-1α/Survivin信号通路的调节关系，并探讨mTOR和HIF-1α/Survivin通路对乏氧A549细胞和H520细胞增殖、侵袭和迁移的影响。
3、探讨榄香烯对乏氧A549和H520细胞的放射增敏作用与mTOR和HIF-1α/Survivin信号通路的关系。
方法：第一部分：榄香烯对乏氧A549和H520细胞的放射增敏作用及对mTOR、HIF-1α和Survivin表达的影响。
1、MTT法检测不同浓度榄香烯对乏氧A549和H520细胞的直接杀伤作用，求出榄香烯作用于乏氧A549和H520细胞24h的IC50值。
2、克隆形成实验观察低浓度的榄香烯对乏氧A549和H520细胞的放射增敏作用。
3、实验分为常氧对照组和乏氧组，根据处理方法不同将乏氧组再分为乏氧对照组、乏氧药物组、乏氧照射组和乏氧联合组。药物作用24h后将乏氧组的细胞放入乏氧盒内乏氧30min，然后于常氧培养箱中培养，2h后取出照射组和联合组细胞给予X线照射。A549细胞和H520细胞经过相应处理后，用RT-PCR和WesternBlot方法检测各组乏氧肺癌细胞中mTOR、HIF-1α、Survivin的表达情况。
4、细胞进行上述相应处理后，用流式细胞仪检测各组A549和H520细胞的凋亡率。
第二部分：mTOR和HIF-1α/Survivin信号通路对乏氧A549和H520细胞增殖、侵袭和迁移活性的影响。
1、将A549和H520细胞分为未转染组、阴性转染组、mTOR siRNA转染组或HIF-1α siRNA转染组，转染6h后，将细胞乏氧30min，然后置于常氧培养箱中继续培养。24h后先检测mTOR和HIF-1α的沉默的效果，然后用RT-PCR和WesternBlot方法检测mTOR表达抑制后肺癌细胞中HIF-1α、Survivin的表达情况，以及HIF-1α表达抑制后Survivin的表达情况。
2、A549和H520细胞分组及处理方法同上，MTT法检测肺癌细胞的吸光度值，分析细胞增殖情况；Transwell小室检测肺癌细胞的侵袭和迁移能力；流式细胞仪检测A549和H520细胞的凋亡率。
第三部分：榄香烯对乏氧A549和H520细胞的放射增敏作用与mTOR和HIF-1α/Survivin信号通路的相关性研究。
1、将A549和H520细胞各分为药物组、照射组、联合组、转染药物组、转染照射组、转染联合组。转染组细胞先用mTOR-siRNA或HIF-1α-siRNA进行细胞转染，6h后所有的药物组和联合组加入20μg/ml的榄香烯作用24h，然后换培养基并将所有组的细胞乏氧30min，于常氧培养箱中继续培养，2h后取出所有的照射组和联合组细胞给予X线照射。24h后用RT-PCR和WesternBlot方法检测mTOR表达抑制后HIF-1α和Survivin的表达，以及HIF-1α表达抑制后Survivin的表达。
2、流式细胞仪检测以上各组A549和H520细胞的凋亡率。
结果：第一部分：
1、MTT法检测不同浓度榄香烯对乏氧A549和H520细胞的直接杀伤作用：榄香烯对乏氧A549和H520细胞均有抑制作用，随着药物浓度的增加，两种细胞的A值逐渐降低，细胞抑制率逐渐增大。在相同药物浓度下，榄香烯对乏氧H520细胞的抑制率大于乏氧A549细胞。榄香烯作用乏氧A549细胞24h的IC50值为112μg/ml，作用乏氧H520细胞24h的IC50值为93μg/ml。
2、克隆形成实验观察榄香烯对乏氧A549和H520细胞的放射增敏作用：在相同照射剂量下，随着药物浓度的增高，乏氧A549和H520细胞存活率逐渐下降（p<0.05）；而在相同药物浓度下，随着照射剂量的逐渐升高，乏氧A549和H520细胞存活率逐渐降低（p<0.05）。低浓度的榄香烯联合照射作用于乏氧A549和H520细胞后，细胞存活率低于单纯照射组（p<0.05）。
3、A549和H520细胞中mTOR、HIF-1α和Survivin的表达：与常氧对照组相比较，乏氧A549细胞中mTOR和HIF-1α的表达均有不同程度的升高，差异有统计学意义（mTOR：p<0.05；HIF-1α：p<0.01）；Survivin的mRNA和蛋白表达也有不同程度升高（蛋白表达：p<0.01；mRNA表达：p>0.05）。H520细胞中，与常氧对照组相比，乏氧对照组的mTOR、HIF-1α和Survivin mRNA表达均升高，差异有统计学意义（p<0.01）。
4、乏氧A549和H520细胞中mTOR表达：与乏氧对照组相比，A549细胞中，乏氧药物组的mTOR mRNA和蛋白表达均有不同程度降低，差异有统计学意义（mRNA：p<0.01；蛋白：p<0.05）。同样与乏氧对照比，乏氧照射组的mRNA和蛋白表达亦有不同程度的降低，差异有统计学意义（mRNA：p<0.01；蛋白：p<0.05）。乏氧联合组的mTOR mRNA和蛋白表达比乏氧药物低，差异有统计意义（mRNA：p<0.01；蛋白p<0.05）。与乏氧照射组相比，乏氧联合组的mTOR mRNA和蛋白表达也有不同程度的降低（mRNA：p<0.01；蛋白p>0.05）。H520细胞中，与乏氧对照组相比，乏氧药物组、照射组的mRNA表达降低，差异有统计学意义（p<0.05）；乏氧联合组的mRNA表达与乏氧药物组、照射比明显降低，差异有统计学意义（p<0.01）。
5、乏氧A549和H520细胞中HIF-1α表达：A549细胞中，乏氧药物组的HIF-1αmRNA和蛋白表达均降低，与乏氧对照组比较差异有统计学意义（p<0.01）。相反，乏氧照射组的HIF-1α mRNA和蛋白表达却有不同程度增高（mRNA表达：p<0.01；蛋白表达：p>0.05）。榄香烯与照射联合可以显著抑制HIF-1α mRNA和蛋白的表达，与乏氧药物组、照射组比较均有统计学意义（p<0.01）。H520细胞中，与乏氧对照组比，乏氧药物组的mRNA表达降低（p<0.01），乏氧照射组的HIF-1α mRNA表达虽然升高，但是没有统计学意义（p>0.05）。榄香烯与照射联合组的HIF-1α mRNA表达低于乏氧药物组、照射组，差异有统计学意义（p<0.01）。
6、乏氧A549和H520细胞中Survivin表达：A549细胞中，与乏氧对照组相比，乏氧药物组的Survivin mRNA和蛋白，以及乏氧照射组的Survivin mRNA表达降低，差异均有统计学意义（p<0.01），虽然乏氧照射组的蛋白表达升高，但差异并没有统计学意义（p>0.05）。联合组的Survivin mRNA和蛋白表达明显低于乏氧药物组、照射组，差异有统计学意义（p<0.01）。H520细胞中，与乏氧对照组相比，乏氧药物组、照射组的Survivin mRNA表达下降，差异有统计学意义（p<0.01）；乏氧联合组Survivin mRNA表达低于乏氧药物组、照射组，差异有统计学意义（p<0.01）。
7、A549和H520细胞凋亡率：A549细胞中，乏氧对照组细胞凋亡率略低于常氧对照组（p>0.05）；与乏氧对照组相比，乏氧药物组、乏氧照射组和乏氧联合组的细胞凋亡率均增高，差异有统计学意义（p<0.01）。在H520细胞中，乏氧对照组细胞凋亡率比常氧对照组略低，差异无统计学意义（p>0.05）；与对照组相比较，乏氧药物组、乏氧照射组和发封邮件联合组的细胞凋亡率均升高，差异有统计学意义（p<0.01）。
第二部分：
1、mTOR和HIF-1α的沉默效果：mTOR siRNA分别转染入A549和H520细胞中后，mTOR表达被有效抑制（p<0.01）。同样，HIF-1α siRNA转染至A549和H520细胞后，HIF-1α的表达也明显降低（p<0.01）。
2、mTOR表达抑制后肺癌细胞中HIF-1α和survivin的表达：与对照组和阴性转染组比较，乏氧A549细胞中HIF-1α和Survivin的mRNA和蛋白表达均降低（p<0.01），乏氧H520细胞中HIF-1α和Survivin的mRNA表达也被抑制（p<0.01）。
3、HIF-1α表达抑制后肺癌细胞中Survivin的表达：与对照组和阴性转染组相比，乏氧A549细胞中Survivin的mRNA和蛋白表达均下降（p<0.01），乏氧H520细胞中的Survivin的mRNA亦有相同趋势（p<0.01）。
4、mTOR或HIF-1α表达抑制后肺癌细胞凋亡率：与对照组相比，mTOR或HIF-1α表达抑制后，乏氧A549和H520细胞的凋亡率均升高（p<0.01）。
5、肺癌细胞增殖活性：mTOR或HIF-1α表达抑制后，乏氧A549和H520细胞的A值比对照组低（p<0.01）。
6、肺癌细胞侵袭和迁移能力：与对照组相比，转染后乏氧A549和H520细胞穿过Transwell小室Matrigel胶或微孔膜的数量明显减少，差异有统计学意义（p<0.01）。
第三部分：
1、mTOR或HIF-1α-siRNA转染至A549和H520细胞中后，药物组、照射组和联合组中mTOR和HIF-1α表达明显降低，与转染前相比差异有统计学意义（p<0.01）。
2、mTOR表达抑制后乏氧肺癌细胞中HIF-1α和Survivin的表达：在乏氧A549细胞中，药物组、照射组和联合组的HIF-1α表达比相应未转染组有不同程度的降低（mRNA:p>0.05；蛋白:p<0.01）；同样，转染后药物组、照射组和联合组的Survivin表达也被明显抑制（p<0.01）。乏氧H520细胞中，与未转染的药物组比较，转染后的药物组HIF-1α mRNA表达略降低，同样转染后的联合组HIF-1α mRNA表达也低于未转染组，差异均无统计学意义（p>0.05），而转染后的照射组HIF-1α mRNA表达与转染前相比明显降低，差异有统计学意义（p<0.01）。与未转染的相应组比较，mTOR表达抑制后的乏氧H520细胞中，药物组、照射组和联合组的SurvivinmRNA表达均降低，差异有统计学意义（p<0.01）。
3、HIF-1α表达抑制后乏氧肺癌细胞中Survivin的表达：在乏氧A549细胞中，药物组、照射组和联合组中的Survivin mRNA和蛋白表达水平均升高，与未转染的相应组比较差异有统计学意义（p<0.01）。在乏氧H520细胞中，同样转染后的药物组、照射组和联合组的Survivin mRNA表达也比相应未转染组高，差异有统计学意义（p<0.01）。
4、肺癌细胞凋亡检测：分别与未转染的药物组、照射组和联合组相比，mTOR或HIF-1α表达抑制后的乏氧A549和H520细胞凋亡率均明显升高（p<0.01）。
结论：第一部分：
1、低浓度的榄香烯对乏氧A549和H520细胞具有放射增敏作用，而且随着药物浓度的增加，增敏作用逐渐增强。
2、乏氧可以诱导A549和H520细胞中mTOR、HIF-1α和Survivin的表达增多，降低细胞凋亡率，提示mTOR、HIF-1α和Survivin与乏氧条件下的肿瘤生存调控有关。
3、榄香烯联合照射可以明显抑制乏氧A549和H520细胞中HIF-1α和Survivin的表达，同时对mTOR表达的抑制比单独榄香烯作用时更明显，提示mTOR、HIF-1α和Survivin可能是榄香烯对乏氧肺癌细胞放射增敏作用的靶点。
4、榄香烯联合照射可以增加乏氧A549细胞和H520细胞的凋亡，提示榄香烯可能通过作用于mTOR、HIF-1α和Survivin而促进乏氧肺癌细胞凋亡。
第二部分：
1、mTOR表达抑制后，乏氧A549和H520细胞的HIF-1α和Survivin的表达减少，细胞凋亡增多；而当HIF-1α表达抑制后，乏氧肺癌细胞中Survivin的表达随之下降，细胞凋亡率同样增高，提示在乏氧A549和H520细胞中，mTOR和HIF-1α/Survivin信号通路之间可能存在着正向调控作用，调节乏氧肺癌细胞的凋亡。
2、mTOR表达抑制以后，乏氧A549和H520细胞的HIF-1α和Survivin的表达减少，细胞增殖、侵袭和迁移力下降。同样当HIF-1α表达抑制后，Survivin的表达下降，肺癌细胞增殖、侵袭和迁移能力也降低，提示乏氧A549和H520细胞活性可能与mTOR和HIF-1α/Survivin通路的调节有关。
第三部分：
1、HIF-1α表达抑制后，榄香烯和照射作用后的乏氧A549和H520细胞中Survivin表达增多，细胞凋亡率升高，提示榄香烯对乏氧肺癌细胞的放射增敏作用可能与HIF-1α/Survivin信号通路的调节有关。
2、mTOR表达抑制后，榄香烯和照射作用后的乏氧A549和H520细胞中HIF-1α和Survivin表达降低，细胞凋亡率增高，提示榄香烯对乏氧肺癌细胞的放射增敏作用可能不是通过作用于mTOR而调节HIF-1α/Survivin的表达，可能还存在有其他的通路抑制HIF-1α/Survivin的表达，进而增加细胞凋亡。
Lung cancer is a malignant tumor threaten to human life worldwide with themortality is1.3million and the non-small-cell lung carcinoma is about80%. Moreadvanced stage patients when diagnosed received modality therapy of radiation andchemotherapy remain the main treatment for them. Researches found that more anoxictumor cells were presented in tumor tissue and the resistances of anoxic cells to radialline caused failure of tumor radiotherapy. To overcome the anoxia condition, peoplesimmediately or indirectly increased oxygen content in tumor tissue but the effectivenesswas also poor. So develop and study tumor anoxic cell radiosensitizer to increase theradiosensitivity become a hot topic in the radiotherapy studies.
Elemene has antitumor effects and also augments the sensitivity to radiotherapyand studies have already confirmed that. Our group discovered that the radiosensitizingeffects of elemene to lung adenocarcinoma A549cell were related with G2/M blocking;up-regulating Bcl-2and down-regulating p53expression resulted to cell apoptosis. Theeffction also related with inhibiting the telomerase activation, inducing DNA damageand inhibiting the repair of DNA damage. Also in vitro study confirmed that elemene incombination with irradiation could improve the tumor anoxic condition to playradiosensitizing effect. Index documents and found that there were few reports about thesignal pass which related with theradiosesiting effects of elemene.
Mammalian target of rapamycin (mTOR), Hypoxia inducible factor1alpha (HIF-α)and Survivin have overexpression in tumors and have close association in rumorigenesisand growth. Also, mTOR and HIF-α are in touch with proliferation, invasion andmigration of tumor cells. If the radiosensitizing effect of elemene has relationship withmTOR and HIF-α/Survivin signal pass, there are few reports about it. This is why to engineer the discussion. The in vivo study of our experimental group detected thatelemene in combination with irradiation could decrease HIF-α and Survivin expressioninduced by irradiation and the inhibition to mTOR was higher than elemene alone, thusto increase the cell apoptosis in tumor. Prompted that mTOR, HIF-α and Survivinmaybe the target points of radiosensitizing effect of elemene. But if it had role mTOR toaffect HIF-α and Survivin was not clearly. The in vitro study of radiosensitizing effectof elemene was under normoxia condition in the past. So in this study, we will detectthe the radiosensitizing effect of elemene to lung adenocarcinoma A549cell andsquamous cell carcinoma H520cell. Also, we probe the effection to mTOR, HIF-1α andSurvivin expression when combine elemene and irradiation to anoxia lung cancer cells.Also, the accommodation of mTOR and HIF-1α/Survivin to cytoactive and the relationto radiosensitizing effect of elemene are under detected.
Objective:
1. Using the light concentration of elemene on anoxia A549and H520cells toapproach the radiosensitizing effect of elemene, and study the influnce of elemene withirradiation to mTOR, HIF-1α and Survivin in anoxic lung cancer A549and H520cells.
2. To validate the regulate relation of mTOR and HIF-1α/Survivin signal pass andobserve the inflrence of the two pathways to A549and H520cell proliferation, invasionand migratory.
3. To investigate the correlation of radiosensitizing effect of elemene to lungcancer cells with mTOR and HIF-1α/Survivin signal pathway.
Methods:
Part one: The influence to mTOR, HIF-1α and Survivin expression ofradiosensitizing effect of elemene to anoxia A549and H520cells.
1. MTT method to detect the lethal effect with different concentration of elemeneto anoxia A549and H520cells and to vent the IC50of A549and H520cells whenelemene effect for24h.
2. Clone formation to observe the radiosensitizing effect of light concentration ofelemene to anoxia A549and H520cells.
3. Grouped A549and H520cells into normoxia control, anoxia control, anoxiaelemene, anoxia irradiation and anoxia combination groups. After20μg/ml β-elemenefor24h, puted the anoxia group cells into anoxia box for30min then cultured innormoxia incubator.Taked out the irradiation and combination groups to receive X-rayirradiation2h later and culture for24h. To detect the expression of mTOR, HIF-1α and Survivin in lung cancer cells using RT-PCR and WseternBlot methods.
4. Gived the same groups and treatments above-mentioned. To detect cellapoptosis of A549and H520by flow cytometer.
Part two: The influence of mTOR and HIF-1α/Survivin signal pathway to anoxiaA549and H520cell proliferation, invasion and migration.
1. Divided A549cells into un-transfect, negative transfect and mTOR-siRNA orHIF-1α-siRNA transfect groups. To detecte the mTOR and HIF-1α expression in A549cell to make sure the silence efficiency by RT-PCR. Then to detecte the HIF-1α andSurvivin expression after silencing mTOR and Survivin expression after silencingHIF-1α using RT-PCR and WesternBlot methods.
2. Divided A549cells into the same groups above-mentioned. MTT method usedto detect the absorbance of lung cancer cells to analysis fell proliferation. Transwellused to detect the ability of invasion and migration of lung caner cells. Flow cytometryused to detect cell apoptosis.
Part three: The correlation of radiosensitizing effect of elemene to A549andH520cell with mTOR and HIF-1α/Survivin signal pathway.
1. Divided A549cells into un-transfect and transfect groups, then divided intothree groups each: elemene, irradiaton and combination groups. The transfect groupsfirst transfect the siRNA into lung cancer cells for6h. After giving the drug with24h, todeal all groups with anoxia and continue to incubation for24h. The cells that needed toirradiate were gived after anoxia. Cells were incubated for24h after theabove-mentioned process, detecting the express of HIF-1α and Survivin after silencingmTOR and the Survivin expression after silencing HIF-1α by RT-PCR andWesternBlot.
2. Detected the cell apoptosis in each group by flow cytometer.
Results:
Part one:1. The different concentration of elemene had the inhibition effect toanoxia A549and H520cells with increasing by the concentration raising. At the sameconcentration, the inhibition to H520cell was higher than A549cell. The IC50of A549cell was112μg/ml and H520cell was93μg/ml.
2. Clone formation to decte the sensetive effect of elemene to lung cancer cells: Atthe same irradiation dose, the cell survival of A549and H520cells were reducing alongwith the dose increasing. The light concentration of elemene with irradiation causedlower cell survival than irradiation alone.
3. The expression of mTOR, HIF-1α and Survivin in A549and H520cells: inA549cells, compared to the normoxia group, the expression of mTOR and HIF-1α wereincreased in different levels and had statistical significance (mTOR: p<0.05; HIF-1α:p<0.01). Survivin mRNA and protein expression were increased in different levels(protein: p<0.01; mRNA: p>0.05). In H520cells, the mTOR, HIF-1α and SurvivinmRNA expression were increased compaired to the normoxia group (p<0.01).
4. The mTOR expression in anoxia A549and H520cells: in A549cell, the mTORmRNA and protein expression in the elemene group and irradiation group wereincreased in different levels (mRNA: p<0.01; protein: p<0.05) contrasted to anoxiacontrol group. The expression of mTOR mRNA and protein in combination group weredecreased in different levels (mRNA: p<0.01; protein: p<0.05) contrasted to elemenegroup. The mTOR mRNA and protein expression in the joint group were also decreasedin different levels (mRNA: p<0.01; protein: p>0.05) compaired to irradiation group. InH520cell, the mTOR mRNA expression in anoxia elemene, irradiation groups werelower than the anoxia control group (p<0.05) and in combination group was decreasedcompaired to elemene and irradiation group (p<0.01).
5. The expression of HIF-1α in anoxia A549and H520cells: in A549cell, theHIF-1α mRNA and protein expression in the elemene group were decreasedsignificantly contrasted to the anoxia group (p<0.01), but the HIF-1α mRNA andprotein expression in irradiation group were increased in defferent levels (mRNA:p<0.01; protein: p>0.05). The HIF-1α mRNA and protein expression in thecombination group were obviously decreased contrasted to elemene and irradiationgroups (p<0.01). In H520cell, compaired to the anoxia control, the HIF-1α mRNAexpression in the anoxia elemene group was decreased (p<0.01) and in anoxiairradiation group was increased (p>0.05). The HIF-1α mRNA expression incombinition group was lower than the elemene and irradiation groups (p<0.01).
6. The expression of Survivin in anoxia A549and H520cells: Contrast to theanoxia control, in A549cell, Survivin mRNA and protein expression in elemene groupand mRNA expression in irradiation were decreased (p<0.01), but the proteinexpression in irradiation group were increased (protein: p>0.05). Survivin mRNA andprotein expression in the combination group were decreased significantly contrasted toelemene and irradiation groups (p<0.01). In H520cell, Survivin mRNA and proteinexpression in elemene and irradiation groups were decreased compaired to anoxiacontrol group (p<0.01) and Survivin mRNA in the combination group was decreased contrasted to elemene and irradiation groups (p<0.01).
7. The cell apoptosis in lung cancer cells: in A549cell, apoptosis in control groupwas higher than normoxia group (p>0.05) and lower than the rest and has statisticalsignificance (p<0.01). The cell apoptosis in combination group was higher thanelemene group and irradiation group (p<0.01). The same tendency was observed inH520cell.
Part two:
1. Screening of mTOR and HIF-1α-siRNA: Transfected mTOR siRNA and HIF-1αsiRNA into A549and H520cells to make sure the gene silence effectivencess. ThemTOR and HIF-1α mRNA expression were both ihibited after transfecting (p<0.01).
2. The expression of HIF-1α and Survivin after transfecting with mTOR siRNA inA549and H520cells: in A549cell, contrasted to control and negative groups, HIF-1αand Survivin mRNA and protein expression were decreased when the mTOR expressionwas inhibited (p<0.01). In H520cell, both HIF-1α and Survivin mRNA expressionwere decreased (p<0.01).
3. The expression of Survivin after transfecting with HIF-1α siRNA in A549andH520cells: in A549cell, compaired to control and negative groups, Survivin mRNAand protein expression were decreased when the HIF-1α expression was inhibited(p<0.01). In H520cell, Survivin mRNA expression had the same tendency compared tocontrol and negative groups (p<0.01).
4. The cell apoptosis of A549and H520cells after transfect: contrasted to controlgroups, the apoptosis were increased in the mTOR or HIF-1α siRNA transfect cells andhad statistical significance (p<0.01).
5. Cell proliferation: Absorbances of A549and H520cells after transfect withmTOR or HIF-1α siRNA were decreased contrasted to control groups (p<0.01).
6. Cell invasion and migeration: The numbers of A549and H520cells passedMatrigel and microporous membrane of Transwell were decreased compared to controlgroups (p<0.01).
Part three:
1. The expression of mTOR and HIF-1α after transfecting with mTOR or HIF-1αsiRNA in lung cancer cells: in A549cell, mTOR and HIF-1α mRNA and proteinexpression were decreased obviously in the transfect groups contrasted to theun-transfect groups (p<0.01). In H520cell, mTOR and HIF-1α mRNA expression werealso decreased compaired to corresp un-transfect groups (p<0.01).
2. The expression of HIF-1α and Survivin after transfecting with mTOR-siRNA inlung cancer cell: in A549cell, the HIF-1α and Survivin mRNA in elemene, irradiationand combination groups were decreased a little in every group (p>0.05) whendown-regulated mTOR, but the HIF-1α protein expression were decreased obviouslyand had statistical significance (p<0.01) compared to corresp un-transfect groups.Survivin mRNA and protein expression in A549cell were decreased compared toun-transfect groups (p<0.01). In H520cell, HIF-1α mRNA expression in elemene andcombination groups were decreased a little (p>0.05) but in irradiation group wasdecreased obviously (p<0.01) contrasted to un-transfect groups. Survivin mRNAexpression in transfect groups of H520cell were down-regulated after mTOR wasdecreased (p<0.01).
3. The expression of Survivin after transfecting with HIF-1α-siRNA in lung cancercells: in A549cell, Survivin mRNA and protein expression in elemene, irradiation andcombination groups were increased (p<0.01) compared to corresp un-transfect groups.In H520cell, Survivin mRNA expression were also increased obviously in everytransfect groups contrasted to corresp un-transfect groups (p<0.01).
4. The cell apoptosis of lung cancer cells: The apoptosis in transfect groups ofA549and H520cells were increased contrast to the corresp un-transfect groups and hadstatistical significance (p<0.01).
Conclusion:
Part one:
1. The light concentration of elemene had radiosensitizing effect to anoxia A549and H520cells and rose up along with the concentration increasing.
2. Hypoxia could increase mTOR, HIF-1α and Survivin expression in differentlevels in A549and H520cells and cell apoptosis were decreased. So improved thatmTOR, HIF-1α and Survivin were correlated with tumor cell survival in anoxiccondiation.
3. Elemene in combination of irradiation could decrease the expression of HIF-1αand Survivin in anoxia A549and H520cells. Meanwhile the inhibition to mTOR wasobviously contrasted to elemene alone. All illustrate that mTOR, HIF-1α and Survivinwere the targets of radiosensitizing effect of elemene.
4. Elemene in combination of irradiation could increase apoptosis of anoxia A549and H520cells. Imply that elemene possible promoted the tumor cell apoptosis througheffecting to mTOR, HIF-1α and Survivin.
Part two:
1. After down-regulating the mTOR expression, the expression of HIF-1α andSurvivin in anoxia A549and H520cells were decreased and cell apoptosis wereincreased. The Survivin expression is decreased when HIF-1α is down-regulated andthe lung cancer cell apoptosis were increased. Prompt that mTOR could influence theactivity of HIF-1α to regulate Survivin expression to induce cell apoptosis.
2. Down-regulation of mTOR could decrease HIF-1α and Survivin expression andSurvivin expression was down-regulated after HIF-1α was inhibated. Both mTOR andHIF-1α was inhibated could reduce the A549and H520cell proliferation, invasion andmigeration. Confirm that mTOR and HIF-1α/Survivin signal pass possibly related withthe regulation of cytoactive of A549and H520cells.
Part three:
1. Elemene and irradiation decreased HIF-1α and Suvivin expression in A549andH520cells after silencing mTOR and cell apoptosis also decreased. Imply that theradiosensitizing effect of elemene possible not through regulating HIF-1α/Suvivinexpression by acting to mTOR. Maybe there were other pathways to regulateHIF-1α/Suvivin expression to induce apoptosis.
2. Survivin levels were increased when Elemene and irradiation effected A549andH520cells after silencing HIF-1α but cell apoptosis were increased. Imply that theradiosensitizing effect of elemene possible correlated with the regulation ofHIF-1α/Survivin.
榄香烯(Elemene)是一种具有抗肿瘤和放射增敏作用的中药，众多基础及临床实验研究也已经证实。课题组前期在研究榄香烯对肺腺癌A549细胞的放射增敏实验中发现，其通过增加肿瘤细胞的G2/M期阻滞、下调Bcl-2和上调p53基因表达促进细胞凋亡而发挥放射增敏作用，其增敏机制也与抑制细胞端粒酶活性、诱导细胞DNA双链和单链损伤及抑制损伤修复有关。体外实验也证实榄香烯联合放疗可以改善肿瘤乏氧状态而发挥放射增敏作用。检索文献发现，对榄香烯放射增敏作用相关的信号通路，研究报道甚少。
哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）、缺氧诱导因子-1α（hypoxia inducible factor-1alpha, HIF-1α）以及生存素（Survivin）在恶性肿瘤组织中有较高表达，与肿瘤细胞的凋亡有着密切联系，而且mTOR和HIF-1α还与恶性肿瘤细胞的增殖、侵袭和转移有关。课题组前期在体内肺癌裸鼠移植瘤实验中发现榄香烯联合放疗可以降低放射诱导的HIF-1α、Survivin表达增多，而且对mTOR的抑制作用比单独榄香烯治疗时强，从而增加移植瘤组织内肺癌细胞的凋亡，提示mTOR、HIF-1α、Survivin可能是榄香烯放射增敏的作用靶点，但是否通过作用于mTOR，进而影响HIF-1α/Survivin的表达，暂时还未明确。以往体外研究榄香烯的放射增敏作用多在常氧条件下进行，因此本实验拟观察榄香烯对乏氧肺腺癌细胞株（A549）和肺鳞癌细胞株（H520）的放射增敏作用，探讨榄香烯联合照射对乏氧肺癌细胞中mTOR、HIF-1α和Survivin表达的影响，以及mTOR和HIF-1α/Survivin通路对肺癌细胞活性的调节作用，和与榄香烯放射增敏作用的关系。
目的：1、选用低浓度榄香烯作用于乏氧A549和H520细胞，观察榄香烯对乏氧肺癌细胞株的放射增敏作用；研究榄香烯联合照射对乏氧A549和H520细胞中mTOR、HIF-1α和Survivin表达的影响。
2、研究mTOR和HIF-1α/Survivin信号通路的调节关系，并探讨mTOR和HIF-1α/Survivin通路对乏氧A549细胞和H520细胞增殖、侵袭和迁移的影响。
3、探讨榄香烯对乏氧A549和H520细胞的放射增敏作用与mTOR和HIF-1α/Survivin信号通路的关系。
方法：第一部分：榄香烯对乏氧A549和H520细胞的放射增敏作用及对mTOR、HIF-1α和Survivin表达的影响。
1、MTT法检测不同浓度榄香烯对乏氧A549和H520细胞的直接杀伤作用，求出榄香烯作用于乏氧A549和H520细胞24h的IC50值。
2、克隆形成实验观察低浓度的榄香烯对乏氧A549和H520细胞的放射增敏作用。
3、实验分为常氧对照组和乏氧组，根据处理方法不同将乏氧组再分为乏氧对照组、乏氧药物组、乏氧照射组和乏氧联合组。药物作用24h后将乏氧组的细胞放入乏氧盒内乏氧30min，然后于常氧培养箱中培养，2h后取出照射组和联合组细胞给予X线照射。A549细胞和H520细胞经过相应处理后，用RT-PCR和WesternBlot方法检测各组乏氧肺癌细胞中mTOR、HIF-1α、Survivin的表达情况。
4、细胞进行上述相应处理后，用流式细胞仪检测各组A549和H520细胞的凋亡率。
第二部分：mTOR和HIF-1α/Survivin信号通路对乏氧A549和H520细胞增殖、侵袭和迁移活性的影响。
1、将A549和H520细胞分为未转染组、阴性转染组、mTOR siRNA转染组或HIF-1α siRNA转染组，转染6h后，将细胞乏氧30min，然后置于常氧培养箱中继续培养。24h后先检测mTOR和HIF-1α的沉默的效果，然后用RT-PCR和WesternBlot方法检测mTOR表达抑制后肺癌细胞中HIF-1α、Survivin的表达情况，以及HIF-1α表达抑制后Survivin的表达情况。
2、A549和H520细胞分组及处理方法同上，MTT法检测肺癌细胞的吸光度值，分析细胞增殖情况；Transwell小室检测肺癌细胞的侵袭和迁移能力；流式细胞仪检测A549和H520细胞的凋亡率。
第三部分：榄香烯对乏氧A549和H520细胞的放射增敏作用与mTOR和HIF-1α/Survivin信号通路的相关性研究。
1、将A549和H520细胞各分为药物组、照射组、联合组、转染药物组、转染照射组、转染联合组。转染组细胞先用mTOR-siRNA或HIF-1α-siRNA进行细胞转染，6h后所有的药物组和联合组加入20μg/ml的榄香烯作用24h，然后换培养基并将所有组的细胞乏氧30min，于常氧培养箱中继续培养，2h后取出所有的照射组和联合组细胞给予X线照射。24h后用RT-PCR和WesternBlot方法检测mTOR表达抑制后HIF-1α和Survivin的表达，以及HIF-1α表达抑制后Survivin的表达。
2、流式细胞仪检测以上各组A549和H520细胞的凋亡率。
结果：第一部分：
1、MTT法检测不同浓度榄香烯对乏氧A549和H520细胞的直接杀伤作用：榄香烯对乏氧A549和H520细胞均有抑制作用，随着药物浓度的增加，两种细胞的A值逐渐降低，细胞抑制率逐渐增大。在相同药物浓度下，榄香烯对乏氧H520细胞的抑制率大于乏氧A549细胞。榄香烯作用乏氧A549细胞24h的IC50值为112μg/ml，作用乏氧H520细胞24h的IC50值为93μg/ml。
2、克隆形成实验观察榄香烯对乏氧A549和H520细胞的放射增敏作用：在相同照射剂量下，随着药物浓度的增高，乏氧A549和H520细胞存活率逐渐下降（p<0.05）；而在相同药物浓度下，随着照射剂量的逐渐升高，乏氧A549和H520细胞存活率逐渐降低（p<0.05）。低浓度的榄香烯联合照射作用于乏氧A549和H520细胞后，细胞存活率低于单纯照射组（p<0.05）。
3、A549和H520细胞中mTOR、HIF-1α和Survivin的表达：与常氧对照组相比较，乏氧A549细胞中mTOR和HIF-1α的表达均有不同程度的升高，差异有统计学意义（mTOR：p<0.05；HIF-1α：p<0.01）；Survivin的mRNA和蛋白表达也有不同程度升高（蛋白表达：p<0.01；mRNA表达：p>0.05）。H520细胞中，与常氧对照组相比，乏氧对照组的mTOR、HIF-1α和Survivin mRNA表达均升高，差异有统计学意义（p<0.01）。
4、乏氧A549和H520细胞中mTOR表达：与乏氧对照组相比，A549细胞中，乏氧药物组的mTOR mRNA和蛋白表达均有不同程度降低，差异有统计学意义（mRNA：p<0.01；蛋白：p<0.05）。同样与乏氧对照比，乏氧照射组的mRNA和蛋白表达亦有不同程度的降低，差异有统计学意义（mRNA：p<0.01；蛋白：p<0.05）。乏氧联合组的mTOR mRNA和蛋白表达比乏氧药物低，差异有统计意义（mRNA：p<0.01；蛋白p<0.05）。与乏氧照射组相比，乏氧联合组的mTOR mRNA和蛋白表达也有不同程度的降低（mRNA：p<0.01；蛋白p>0.05）。H520细胞中，与乏氧对照组相比，乏氧药物组、照射组的mRNA表达降低，差异有统计学意义（p<0.05）；乏氧联合组的mRNA表达与乏氧药物组、照射比明显降低，差异有统计学意义（p<0.01）。
5、乏氧A549和H520细胞中HIF-1α表达：A549细胞中，乏氧药物组的HIF-1αmRNA和蛋白表达均降低，与乏氧对照组比较差异有统计学意义（p<0.01）。相反，乏氧照射组的HIF-1α mRNA和蛋白表达却有不同程度增高（mRNA表达：p<0.01；蛋白表达：p>0.05）。榄香烯与照射联合可以显著抑制HIF-1α mRNA和蛋白的表达，与乏氧药物组、照射组比较均有统计学意义（p<0.01）。H520细胞中，与乏氧对照组比，乏氧药物组的mRNA表达降低（p<0.01），乏氧照射组的HIF-1α mRNA表达虽然升高，但是没有统计学意义（p>0.05）。榄香烯与照射联合组的HIF-1α mRNA表达低于乏氧药物组、照射组，差异有统计学意义（p<0.01）。
6、乏氧A549和H520细胞中Survivin表达：A549细胞中，与乏氧对照组相比，乏氧药物组的Survivin mRNA和蛋白，以及乏氧照射组的Survivin mRNA表达降低，差异均有统计学意义（p<0.01），虽然乏氧照射组的蛋白表达升高，但差异并没有统计学意义（p>0.05）。联合组的Survivin mRNA和蛋白表达明显低于乏氧药物组、照射组，差异有统计学意义（p<0.01）。H520细胞中，与乏氧对照组相比，乏氧药物组、照射组的Survivin mRNA表达下降，差异有统计学意义（p<0.01）；乏氧联合组Survivin mRNA表达低于乏氧药物组、照射组，差异有统计学意义（p<0.01）。
7、A549和H520细胞凋亡率：A549细胞中，乏氧对照组细胞凋亡率略低于常氧对照组（p>0.05）；与乏氧对照组相比，乏氧药物组、乏氧照射组和乏氧联合组的细胞凋亡率均增高，差异有统计学意义（p<0.01）。在H520细胞中，乏氧对照组细胞凋亡率比常氧对照组略低，差异无统计学意义（p>0.05）；与对照组相比较，乏氧药物组、乏氧照射组和发封邮件联合组的细胞凋亡率均升高，差异有统计学意义（p<0.01）。
第二部分：
1、mTOR和HIF-1α的沉默效果：mTOR siRNA分别转染入A549和H520细胞中后，mTOR表达被有效抑制（p<0.01）。同样，HIF-1α siRNA转染至A549和H520细胞后，HIF-1α的表达也明显降低（p<0.01）。
2、mTOR表达抑制后肺癌细胞中HIF-1α和survivin的表达：与对照组和阴性转染组比较，乏氧A549细胞中HIF-1α和Survivin的mRNA和蛋白表达均降低（p<0.01），乏氧H520细胞中HIF-1α和Survivin的mRNA表达也被抑制（p<0.01）。
3、HIF-1α表达抑制后肺癌细胞中Survivin的表达：与对照组和阴性转染组相比，乏氧A549细胞中Survivin的mRNA和蛋白表达均下降（p<0.01），乏氧H520细胞中的Survivin的mRNA亦有相同趋势（p<0.01）。
4、mTOR或HIF-1α表达抑制后肺癌细胞凋亡率：与对照组相比，mTOR或HIF-1α表达抑制后，乏氧A549和H520细胞的凋亡率均升高（p<0.01）。
5、肺癌细胞增殖活性：mTOR或HIF-1α表达抑制后，乏氧A549和H520细胞的A值比对照组低（p<0.01）。
6、肺癌细胞侵袭和迁移能力：与对照组相比，转染后乏氧A549和H520细胞穿过Transwell小室Matrigel胶或微孔膜的数量明显减少，差异有统计学意义（p<0.01）。
第三部分：
1、mTOR或HIF-1α-siRNA转染至A549和H520细胞中后，药物组、照射组和联合组中mTOR和HIF-1α表达明显降低，与转染前相比差异有统计学意义（p<0.01）。
2、mTOR表达抑制后乏氧肺癌细胞中HIF-1α和Survivin的表达：在乏氧A549细胞中，药物组、照射组和联合组的HIF-1α表达比相应未转染组有不同程度的降低（mRNA:p>0.05；蛋白:p<0.01）；同样，转染后药物组、照射组和联合组的Survivin表达也被明显抑制（p<0.01）。乏氧H520细胞中，与未转染的药物组比较，转染后的药物组HIF-1α mRNA表达略降低，同样转染后的联合组HIF-1α mRNA表达也低于未转染组，差异均无统计学意义（p>0.05），而转染后的照射组HIF-1α mRNA表达与转染前相比明显降低，差异有统计学意义（p<0.01）。与未转染的相应组比较，mTOR表达抑制后的乏氧H520细胞中，药物组、照射组和联合组的SurvivinmRNA表达均降低，差异有统计学意义（p<0.01）。
3、HIF-1α表达抑制后乏氧肺癌细胞中Survivin的表达：在乏氧A549细胞中，药物组、照射组和联合组中的Survivin mRNA和蛋白表达水平均升高，与未转染的相应组比较差异有统计学意义（p<0.01）。在乏氧H520细胞中，同样转染后的药物组、照射组和联合组的Survivin mRNA表达也比相应未转染组高，差异有统计学意义（p<0.01）。
4、肺癌细胞凋亡检测：分别与未转染的药物组、照射组和联合组相比，mTOR或HIF-1α表达抑制后的乏氧A549和H520细胞凋亡率均明显升高（p<0.01）。
结论：第一部分：
1、低浓度的榄香烯对乏氧A549和H520细胞具有放射增敏作用，而且随着药物浓度的增加，增敏作用逐渐增强。
2、乏氧可以诱导A549和H520细胞中mTOR、HIF-1α和Survivin的表达增多，降低细胞凋亡率，提示mTOR、HIF-1α和Survivin与乏氧条件下的肿瘤生存调控有关。
3、榄香烯联合照射可以明显抑制乏氧A549和H520细胞中HIF-1α和Survivin的表达，同时对mTOR表达的抑制比单独榄香烯作用时更明显，提示mTOR、HIF-1α和Survivin可能是榄香烯对乏氧肺癌细胞放射增敏作用的靶点。
4、榄香烯联合照射可以增加乏氧A549细胞和H520细胞的凋亡，提示榄香烯可能通过作用于mTOR、HIF-1α和Survivin而促进乏氧肺癌细胞凋亡。
第二部分：
1、mTOR表达抑制后，乏氧A549和H520细胞的HIF-1α和Survivin的表达减少，细胞凋亡增多；而当HIF-1α表达抑制后，乏氧肺癌细胞中Survivin的表达随之下降，细胞凋亡率同样增高，提示在乏氧A549和H520细胞中，mTOR和HIF-1α/Survivin信号通路之间可能存在着正向调控作用，调节乏氧肺癌细胞的凋亡。
2、mTOR表达抑制以后，乏氧A549和H520细胞的HIF-1α和Survivin的表达减少，细胞增殖、侵袭和迁移力下降。同样当HIF-1α表达抑制后，Survivin的表达下降，肺癌细胞增殖、侵袭和迁移能力也降低，提示乏氧A549和H520细胞活性可能与mTOR和HIF-1α/Survivin通路的调节有关。
第三部分：
1、HIF-1α表达抑制后，榄香烯和照射作用后的乏氧A549和H520细胞中Survivin表达增多，细胞凋亡率升高，提示榄香烯对乏氧肺癌细胞的放射增敏作用可能与HIF-1α/Survivin信号通路的调节有关。
2、mTOR表达抑制后，榄香烯和照射作用后的乏氧A549和H520细胞中HIF-1α和Survivin表达降低，细胞凋亡率增高，提示榄香烯对乏氧肺癌细胞的放射增敏作用可能不是通过作用于mTOR而调节HIF-1α/Survivin的表达，可能还存在有其他的通路抑制HIF-1α/Survivin的表达，进而增加细胞凋亡。
Lung cancer is a malignant tumor threaten to human life worldwide with themortality is1.3million and the non-small-cell lung carcinoma is about80%. Moreadvanced stage patients when diagnosed received modality therapy of radiation andchemotherapy remain the main treatment for them. Researches found that more anoxictumor cells were presented in tumor tissue and the resistances of anoxic cells to radialline caused failure of tumor radiotherapy. To overcome the anoxia condition, peoplesimmediately or indirectly increased oxygen content in tumor tissue but the effectivenesswas also poor. So develop and study tumor anoxic cell radiosensitizer to increase theradiosensitivity become a hot topic in the radiotherapy studies.
Elemene has antitumor effects and also augments the sensitivity to radiotherapyand studies have already confirmed that. Our group discovered that the radiosensitizingeffects of elemene to lung adenocarcinoma A549cell were related with G2/M blocking;up-regulating Bcl-2and down-regulating p53expression resulted to cell apoptosis. Theeffction also related with inhibiting the telomerase activation, inducing DNA damageand inhibiting the repair of DNA damage. Also in vitro study confirmed that elemene incombination with irradiation could improve the tumor anoxic condition to playradiosensitizing effect. Index documents and found that there were few reports about thesignal pass which related with theradiosesiting effects of elemene.
Mammalian target of rapamycin (mTOR), Hypoxia inducible factor1alpha (HIF-α)and Survivin have overexpression in tumors and have close association in rumorigenesisand growth. Also, mTOR and HIF-α are in touch with proliferation, invasion andmigration of tumor cells. If the radiosensitizing effect of elemene has relationship withmTOR and HIF-α/Survivin signal pass, there are few reports about it. This is why to engineer the discussion. The in vivo study of our experimental group detected thatelemene in combination with irradiation could decrease HIF-α and Survivin expressioninduced by irradiation and the inhibition to mTOR was higher than elemene alone, thusto increase the cell apoptosis in tumor. Prompted that mTOR, HIF-α and Survivinmaybe the target points of radiosensitizing effect of elemene. But if it had role mTOR toaffect HIF-α and Survivin was not clearly. The in vitro study of radiosensitizing effectof elemene was under normoxia condition in the past. So in this study, we will detectthe the radiosensitizing effect of elemene to lung adenocarcinoma A549cell andsquamous cell carcinoma H520cell. Also, we probe the effection to mTOR, HIF-1α andSurvivin expression when combine elemene and irradiation to anoxia lung cancer cells.Also, the accommodation of mTOR and HIF-1α/Survivin to cytoactive and the relationto radiosensitizing effect of elemene are under detected.
Objective:
1. Using the light concentration of elemene on anoxia A549and H520cells toapproach the radiosensitizing effect of elemene, and study the influnce of elemene withirradiation to mTOR, HIF-1α and Survivin in anoxic lung cancer A549and H520cells.
2. To validate the regulate relation of mTOR and HIF-1α/Survivin signal pass andobserve the inflrence of the two pathways to A549and H520cell proliferation, invasionand migratory.
3. To investigate the correlation of radiosensitizing effect of elemene to lungcancer cells with mTOR and HIF-1α/Survivin signal pathway.
Methods:
Part one: The influence to mTOR, HIF-1α and Survivin expression ofradiosensitizing effect of elemene to anoxia A549and H520cells.
1. MTT method to detect the lethal effect with different concentration of elemeneto anoxia A549and H520cells and to vent the IC50of A549and H520cells whenelemene effect for24h.
2. Clone formation to observe the radiosensitizing effect of light concentration ofelemene to anoxia A549and H520cells.
3. Grouped A549and H520cells into normoxia control, anoxia control, anoxiaelemene, anoxia irradiation and anoxia combination groups. After20μg/ml β-elemenefor24h, puted the anoxia group cells into anoxia box for30min then cultured innormoxia incubator.Taked out the irradiation and combination groups to receive X-rayirradiation2h later and culture for24h. To detect the expression of mTOR, HIF-1α and Survivin in lung cancer cells using RT-PCR and WseternBlot methods.
4. Gived the same groups and treatments above-mentioned. To detect cellapoptosis of A549and H520by flow cytometer.
Part two: The influence of mTOR and HIF-1α/Survivin signal pathway to anoxiaA549and H520cell proliferation, invasion and migration.
1. Divided A549cells into un-transfect, negative transfect and mTOR-siRNA orHIF-1α-siRNA transfect groups. To detecte the mTOR and HIF-1α expression in A549cell to make sure the silence efficiency by RT-PCR. Then to detecte the HIF-1α andSurvivin expression after silencing mTOR and Survivin expression after silencingHIF-1α using RT-PCR and WesternBlot methods.
2. Divided A549cells into the same groups above-mentioned. MTT method usedto detect the absorbance of lung cancer cells to analysis fell proliferation. Transwellused to detect the ability of invasion and migration of lung caner cells. Flow cytometryused to detect cell apoptosis.
Part three: The correlation of radiosensitizing effect of elemene to A549andH520cell with mTOR and HIF-1α/Survivin signal pathway.
1. Divided A549cells into un-transfect and transfect groups, then divided intothree groups each: elemene, irradiaton and combination groups. The transfect groupsfirst transfect the siRNA into lung cancer cells for6h. After giving the drug with24h, todeal all groups with anoxia and continue to incubation for24h. The cells that needed toirradiate were gived after anoxia. Cells were incubated for24h after theabove-mentioned process, detecting the express of HIF-1α and Survivin after silencingmTOR and the Survivin expression after silencing HIF-1α by RT-PCR andWesternBlot.
2. Detected the cell apoptosis in each group by flow cytometer.
Results:
Part one:1. The different concentration of elemene had the inhibition effect toanoxia A549and H520cells with increasing by the concentration raising. At the sameconcentration, the inhibition to H520cell was higher than A549cell. The IC50of A549cell was112μg/ml and H520cell was93μg/ml.
2. Clone formation to decte the sensetive effect of elemene to lung cancer cells: Atthe same irradiation dose, the cell survival of A549and H520cells were reducing alongwith the dose increasing. The light concentration of elemene with irradiation causedlower cell survival than irradiation alone.
3. The expression of mTOR, HIF-1α and Survivin in A549and H520cells: inA549cells, compared to the normoxia group, the expression of mTOR and HIF-1α wereincreased in different levels and had statistical significance (mTOR: p<0.05; HIF-1α:p<0.01). Survivin mRNA and protein expression were increased in different levels(protein: p<0.01; mRNA: p>0.05). In H520cells, the mTOR, HIF-1α and SurvivinmRNA expression were increased compaired to the normoxia group (p<0.01).
4. The mTOR expression in anoxia A549and H520cells: in A549cell, the mTORmRNA and protein expression in the elemene group and irradiation group wereincreased in different levels (mRNA: p<0.01; protein: p<0.05) contrasted to anoxiacontrol group. The expression of mTOR mRNA and protein in combination group weredecreased in different levels (mRNA: p<0.01; protein: p<0.05) contrasted to elemenegroup. The mTOR mRNA and protein expression in the joint group were also decreasedin different levels (mRNA: p<0.01; protein: p>0.05) compaired to irradiation group. InH520cell, the mTOR mRNA expression in anoxia elemene, irradiation groups werelower than the anoxia control group (p<0.05) and in combination group was decreasedcompaired to elemene and irradiation group (p<0.01).
5. The expression of HIF-1α in anoxia A549and H520cells: in A549cell, theHIF-1α mRNA and protein expression in the elemene group were decreasedsignificantly contrasted to the anoxia group (p<0.01), but the HIF-1α mRNA andprotein expression in irradiation group were increased in defferent levels (mRNA:p<0.01; protein: p>0.05). The HIF-1α mRNA and protein expression in thecombination group were obviously decreased contrasted to elemene and irradiationgroups (p<0.01). In H520cell, compaired to the anoxia control, the HIF-1α mRNAexpression in the anoxia elemene group was decreased (p<0.01) and in anoxiairradiation group was increased (p>0.05). The HIF-1α mRNA expression incombinition group was lower than the elemene and irradiation groups (p<0.01).
6. The expression of Survivin in anoxia A549and H520cells: Contrast to theanoxia control, in A549cell, Survivin mRNA and protein expression in elemene groupand mRNA expression in irradiation were decreased (p<0.01), but the proteinexpression in irradiation group were increased (protein: p>0.05). Survivin mRNA andprotein expression in the combination group were decreased significantly contrasted toelemene and irradiation groups (p<0.01). In H520cell, Survivin mRNA and proteinexpression in elemene and irradiation groups were decreased compaired to anoxiacontrol group (p<0.01) and Survivin mRNA in the combination group was decreased contrasted to elemene and irradiation groups (p<0.01).
7. The cell apoptosis in lung cancer cells: in A549cell, apoptosis in control groupwas higher than normoxia group (p>0.05) and lower than the rest and has statisticalsignificance (p<0.01). The cell apoptosis in combination group was higher thanelemene group and irradiation group (p<0.01). The same tendency was observed inH520cell.
Part two:
1. Screening of mTOR and HIF-1α-siRNA: Transfected mTOR siRNA and HIF-1αsiRNA into A549and H520cells to make sure the gene silence effectivencess. ThemTOR and HIF-1α mRNA expression were both ihibited after transfecting (p<0.01).
2. The expression of HIF-1α and Survivin after transfecting with mTOR siRNA inA549and H520cells: in A549cell, contrasted to control and negative groups, HIF-1αand Survivin mRNA and protein expression were decreased when the mTOR expressionwas inhibited (p<0.01). In H520cell, both HIF-1α and Survivin mRNA expressionwere decreased (p<0.01).
3. The expression of Survivin after transfecting with HIF-1α siRNA in A549andH520cells: in A549cell, compaired to control and negative groups, Survivin mRNAand protein expression were decreased when the HIF-1α expression was inhibited(p<0.01). In H520cell, Survivin mRNA expression had the same tendency compared tocontrol and negative groups (p<0.01).
4. The cell apoptosis of A549and H520cells after transfect: contrasted to controlgroups, the apoptosis were increased in the mTOR or HIF-1α siRNA transfect cells andhad statistical significance (p<0.01).
5. Cell proliferation: Absorbances of A549and H520cells after transfect withmTOR or HIF-1α siRNA were decreased contrasted to control groups (p<0.01).
6. Cell invasion and migeration: The numbers of A549and H520cells passedMatrigel and microporous membrane of Transwell were decreased compared to controlgroups (p<0.01).
Part three:
1. The expression of mTOR and HIF-1α after transfecting with mTOR or HIF-1αsiRNA in lung cancer cells: in A549cell, mTOR and HIF-1α mRNA and proteinexpression were decreased obviously in the transfect groups contrasted to theun-transfect groups (p<0.01). In H520cell, mTOR and HIF-1α mRNA expression werealso decreased compaired to corresp un-transfect groups (p<0.01).
2. The expression of HIF-1α and Survivin after transfecting with mTOR-siRNA inlung cancer cell: in A549cell, the HIF-1α and Survivin mRNA in elemene, irradiationand combination groups were decreased a little in every group (p>0.05) whendown-regulated mTOR, but the HIF-1α protein expression were decreased obviouslyand had statistical significance (p<0.01) compared to corresp un-transfect groups.Survivin mRNA and protein expression in A549cell were decreased compared toun-transfect groups (p<0.01). In H520cell, HIF-1α mRNA expression in elemene andcombination groups were decreased a little (p>0.05) but in irradiation group wasdecreased obviously (p<0.01) contrasted to un-transfect groups. Survivin mRNAexpression in transfect groups of H520cell were down-regulated after mTOR wasdecreased (p<0.01).
3. The expression of Survivin after transfecting with HIF-1α-siRNA in lung cancercells: in A549cell, Survivin mRNA and protein expression in elemene, irradiation andcombination groups were increased (p<0.01) compared to corresp un-transfect groups.In H520cell, Survivin mRNA expression were also increased obviously in everytransfect groups contrasted to corresp un-transfect groups (p<0.01).
4. The cell apoptosis of lung cancer cells: The apoptosis in transfect groups ofA549and H520cells were increased contrast to the corresp un-transfect groups and hadstatistical significance (p<0.01).
Conclusion:
Part one:
1. The light concentration of elemene had radiosensitizing effect to anoxia A549and H520cells and rose up along with the concentration increasing.
2. Hypoxia could increase mTOR, HIF-1α and Survivin expression in differentlevels in A549and H520cells and cell apoptosis were decreased. So improved thatmTOR, HIF-1α and Survivin were correlated with tumor cell survival in anoxiccondiation.
3. Elemene in combination of irradiation could decrease the expression of HIF-1αand Survivin in anoxia A549and H520cells. Meanwhile the inhibition to mTOR wasobviously contrasted to elemene alone. All illustrate that mTOR, HIF-1α and Survivinwere the targets of radiosensitizing effect of elemene.
4. Elemene in combination of irradiation could increase apoptosis of anoxia A549and H520cells. Imply that elemene possible promoted the tumor cell apoptosis througheffecting to mTOR, HIF-1α and Survivin.
Part two:
1. After down-regulating the mTOR expression, the expression of HIF-1α andSurvivin in anoxia A549and H520cells were decreased and cell apoptosis wereincreased. The Survivin expression is decreased when HIF-1α is down-regulated andthe lung cancer cell apoptosis were increased. Prompt that mTOR could influence theactivity of HIF-1α to regulate Survivin expression to induce cell apoptosis.
2. Down-regulation of mTOR could decrease HIF-1α and Survivin expression andSurvivin expression was down-regulated after HIF-1α was inhibated. Both mTOR andHIF-1α was inhibated could reduce the A549and H520cell proliferation, invasion andmigeration. Confirm that mTOR and HIF-1α/Survivin signal pass possibly related withthe regulation of cytoactive of A549and H520cells.
Part three:
1. Elemene and irradiation decreased HIF-1α and Suvivin expression in A549andH520cells after silencing mTOR and cell apoptosis also decreased. Imply that theradiosensitizing effect of elemene possible not through regulating HIF-1α/Suvivinexpression by acting to mTOR. Maybe there were other pathways to regulateHIF-1α/Suvivin expression to induce apoptosis.
2. Survivin levels were increased when Elemene and irradiation effected A549andH520cells after silencing HIF-1α but cell apoptosis were increased. Imply that theradiosensitizing effect of elemene possible correlated with the regulation ofHIF-1α/Survivin.
引文
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