DATS和辛伐他汀对骨肉瘤细胞的作用及机制研究
发布时间：2018-08-07 22:03
【摘要】：第一部分DATS对骨肉瘤细胞的作用及机制研究研究背景骨肉瘤是最常见的起源于骨骼系统的原发性恶性骨肿瘤,主要发生于儿童和青少年中。肺转移是骨肉瘤最常见的早期转移部位,也是导致死亡的重要原因。目前骨肉瘤的标准治疗方式是术前的新辅助化疗,手术切除,术后辅助化疗。然而,即使采用了广泛的外科手术切除及高强度的辅助化疗方案,大约35-55%的初诊局限肿瘤患者会出现肿瘤复发或转移。而化疗耐药的出现和化疗的副作用进一步影响了骨肉瘤治疗的效果。因此,需要继续开发新的治疗药物来进一步提高骨肉瘤治疗的效果并改善预后。大蒜素是从大蒜的鳞茎中提取的天然有机硫化物(organosulfur compounds, OSCs),是多种具有挥发性的烯丙基硫化物的混合物,主要成分包括二烯丙基一硫化物(diallyl sulfide, DAS)、二烯丙基二硫化物(diallyl disulfide, DADS)、二烯丙基三硫化物(diallyl trisulfide, DATS)和大蒜烯(Ajoene)。近年来,流行病学研究中发现大蒜的摄入量与某些肿瘤的发生呈负相关,很多体内和体外实验也证实了大蒜素具有明显的抗肿瘤活性。在大蒜素的各种活性成分中,DATS显示出了相比其他几种成分更强的抗肿瘤特性,是一种潜在的抗癌药物。既往研究显示DATS对胃癌、结肠癌、肺癌、乳腺癌、前列腺癌具有明显得抗肿瘤作用。然而,DATS对骨肉瘤的作用及机制研究尚不充分。本研究中我们应用骨肉瘤细胞系MG63和MNNG/HOS两种细胞作为研究对象,研究DATS对两种细胞的作用及可能涉及的分子机制,以期为DATS用于治疗骨肉瘤提供了理论依据。研究目的1.观察DATS对骨肉瘤细胞的作用。2.探寻DATS抑制骨肉瘤生长的可能作用机制。研究方法1.应用不同浓度的DATS处理骨肉瘤细胞MG63和MNNG/HOS 24h,48h,72h后,用CCK-8法检测骨肉瘤细胞细胞活性,分析DATS对骨肉瘤细胞增殖的影响。2.应用不同浓度DATS孵育MG63和MNNG/HOS细胞48h后,倒置显微镜下观察细胞的形态学变化,观察DATS对骨肉瘤细胞的形态学影响。3.应用细胞周期检测试剂盒检测DATS处理骨肉瘤细胞MG63和MNNG/HOS后的细胞周期分布变化,观察DATS对骨肉瘤细胞周期分布的影响。应用Western Blot检测细胞周期相关蛋白cyclin D1、p21、p27的表达,分析DATS诱导骨肉瘤细胞G0/G1周期阻滞的可能分子机制。4.应用细胞凋亡检测试剂盒检测DATS处理骨肉瘤细胞MG63和MNNG/HOS后的细胞细胞凋亡比率,观察DATS对骨肉瘤细胞凋亡的影响。5.应用荧光探针DCFH-DA通过荧光显微镜和流式细胞仪检测DATS诱导MG63和MNNG/HOS细胞内活性氧(ROS)变化水平。6.应用荧光探针JC-1检测DATS处理后的骨肉瘤细胞MG63和MNNG/HOS的线粒体膜电位变化。7. DATS处理MG63和MNNG/HOS细胞后,提取总蛋白,应用Western Blot检测PI3K/Akt信号通路中PI3Kp110β、PI3Kp85α、Akt、phosoho-Akt的表达及下游分子Bad、Bax、Bcl-2、Bcl-XL的表达,检测线粒体凋亡通路中cytochrome c、caspase-9、caspase-3、cleaved PARP蛋白表达变化。分析DATS诱导骨肉瘤细胞凋亡的可能分子机制。实验结果1. DATS对骨肉瘤细胞MG63和MNNG/HOS的活性抑制作用随药物浓度和处理时间的增加而增强。DATS对MG63细胞活性有着更强的抑制作用。2. DATS处理细胞48h后,导致骨肉瘤细胞发生明显的形态变化。3. DATS诱导MG63和MNNG/HOS细胞发生G0/G1周期阻滞,抗氧化剂NAC能够逆转DATS诱导的G0/G1周期阻滞。DATS处理骨肉瘤细胞后cyclin D1蛋白表达下调,而p21和p27蛋白的表达明显上调。4. DATS能以剂量和时间依赖的方式诱导骨肉瘤细胞发生凋亡。NAC预处理细胞后可逆转DATS诱导的凋亡。5. DATS作用MG63和MNNG/HOS 16h后,细胞内ROS水平明显增加。DATS随着浓度增加和作用时间延长而明显增加骨肉瘤细胞内ROS水平。NAC则完全消除了ROS的增加。6. DATS作用骨肉瘤细胞后,线粒体膜电位水平发生明显下降,NAC对抗DATS对线粒体膜电位的破坏作用。7. DATS作用后骨肉瘤细胞内PI3Kp11oβ, PI3Kp85a, Akt和p-Akt的蛋白表达呈现与剂量相关的下降。同时Bad和Bax表达上调,而Bcl-2和Bcl-XL表达下调,细胞内cytochrome c及活化的caspase-9和caspase-3水平,cleaved PARP蛋白表达明显增加。说明DATS通过下调PI3K/Akt通路和激活线粒体凋亡通路诱导骨肉瘤细胞凋亡。同时,DATS对PI3K/Akt信号通路起到了PI3K/Akt信号通路抑制剂LY294002的类似作用,而LY294002和DATS共同处理细胞时起到了明显的协同抑制作用。NAC完全逆转DATS对PI3K/Akt通路蛋白的下调,说明DATS诱导的骨肉瘤细胞MG63和MNNG/HOS凋亡是依赖于ROS增加导致的PI3K/Akt信号通路下调起作用的。结论1. DATS具有明显的抑制骨肉瘤细胞生长的作用。2. DATS通过增加ROS的产生及下调cyclin D1、上调p21和p27蛋白表达而诱导骨肉瘤细胞G0/G1周期阻滞。3. DATS通过诱导ROS产生导致骨肉瘤细胞线粒体膜电位水平下降。4. DATS通过增加ROS增加诱导骨肉瘤细胞凋亡,其分子机制可能与ROS依赖的PI3K/Akt信号通路下调及线粒体凋亡通路活化有关。第二部分辛伐他汀对骨肉瘤细胞的作用及机制研究研究背景骨肉瘤是主要发生于青少年人群的最常见的原发性恶性骨肿瘤,是青少年肿瘤致死的主要原因之一。即使采用了广泛的外科手术切除及新辅助化疗方案,目前骨肉瘤的治疗效果仍然较差,5年生存率在过去二十年来并未得到明显的提高。因此,研究或开发新的治疗骨肉瘤的有效药物迫在眉睫。他汀类药物是羟甲基戊二酸单酰辅酶A (3-hydroxy-3-methylglutaryl coenzyme A, HMG-CoA)还原酶的竞争性抑制剂,而HMG-CoA还原酶是人体内内源性胆固醇合成的限速酶。他汀类药物通过抑制HMG-CoA还原酶的活性,能够有效的减少细胞内胆固醇的合成,从而降低血液中的胆固醇水平。近阶段以来,他汀类药物的抗肿瘤作用获得了越来越多研究者的注意,并且被证实能够抑制多种肿瘤的生长,促进其凋亡。在诸多的他汀类药物中,辛伐他汀在体内、体外和动物实验中显示出了相较其他同类药物更强的抗肿瘤特性。之前的体外研究证实,辛伐他汀能有效的抑制卵巢癌、胆管癌、肾癌、前列腺癌和肝癌的生长,其主要的抗肿瘤机制是诱导凋亡及周期阻滞。然而,辛伐他汀对骨肉瘤的作用目前研究较少,其抗骨肉瘤效果及机制文献报道不多。本研究将通过一系列方法检测辛伐他汀对骨肉瘤的作用,并探寻可能涉及的分子机制,为辛伐他汀治疗骨肉瘤的可能前景提供理论依据。研究目的1.观察辛伐他汀对骨肉瘤细胞的作用。2.探寻辛伐他汀抑制骨肉瘤生长的可能信号通路及分子机制。研究方法1.应用不同浓度的辛伐他汀处理骨肉瘤细胞MNNG/HOS 24h,48h,72h后,用CCK-8法检测骨肉瘤细胞细胞活性,分析辛伐他汀对骨肉瘤细胞增殖的影响。2.应用细胞划痕实验(愈伤实验)检测辛伐他汀处理骨肉瘤细胞MNNG/HOS后的细胞迁移距离,观察辛伐他汀对骨肉瘤细胞迁移能力影响。3.应用底面铺有Matrigel基质胶的Transwell小室检测辛伐他汀对骨肉瘤细胞侵袭能力的影响。应用Western Blot检测基质金属蛋白酶(MMPs) MMP-2、MMP-9的蛋白表达,分析辛伐他汀抑制抑制骨肉瘤细胞迁移侵袭的可能分子机制。4.应用细胞周期检测试剂盒检测辛伐他汀处理骨肉瘤细胞MNNG/HOS后的细胞周期分布变化,观察辛伐他汀对骨肉瘤细胞周期分布的影响。应用Western Blot检测细胞周期相关蛋白cyclin D1、CDK2、CDK4、p21、p27的表达,分析辛伐他汀诱导骨肉瘤细胞GO/G1周期阻滞的可能分子机制。5.应用细胞凋亡检测试剂盒检测辛伐他汀对骨肉瘤细胞凋亡的影响。应用Western Blot检测PI3K/Akt信号通路中PI3K、Akt、p-Akt的表达及下游的Bax、Bcl-2、cleaved PARP蛋白表达变化。分析辛伐他汀诱导骨肉瘤细胞凋亡的可能分子机制。6.应用PI3K/Akt信号通路激活剂IGF-1和特异性抑制剂LY294002及不同浓度的辛伐他汀分别或者共同作用MNNG/HOS细胞后,提取总蛋白,检测PI3K、Akt、p-Akt的表达及下游的Bax、Bcl-2、cleaved PARP蛋白表达变化,分析PI3K/Akt信号通路在辛伐他汀诱导骨肉瘤细胞凋亡中的作用。7.建立骨肉瘤MNNG/HOS细胞的动物模型,给予腹腔注射辛伐他汀或生理盐水,定期称量裸鼠体重,测量计算移植瘤的体积。观察辛伐他汀对动物模型中骨肉瘤的生长抑制情况。实验结果1.辛伐他汀以时间和浓度依赖方式抑制骨肉瘤细胞MNNG/HOS的增殖。2.辛伐他汀抑制骨肉瘤细胞MNNG/HOS的迁移,并呈剂量依赖性。3.辛伐他汀抑制骨肉瘤细胞MNNG/HOS的侵袭,并呈剂量依赖性。同时,MNNG/HOS细胞中的MMP-2和MMP-9蛋白的表达明显下降,说明辛伐他汀抑制骨肉瘤细胞侵袭的分子机制可能与下调MMP-2和MMP-9蛋白的表达有关。4.辛伐他汀诱导MNNG/HOS细胞G0/G1周期阻滞。Western Blot结果显示,细胞内cyclin D1、CDK2、CDK4蛋白的表达明显下调,而p21和p27蛋白的表达则被明显上调,这个结果解释了辛伐他汀诱导骨肉瘤细胞G0/G1周期阻滞的可能分子机制。5.辛伐他汀作用后,MNNG/HOS细胞凋亡比率明显增加并与剂量正相关。Western blot结果显示,辛伐他汀作用后PI3K和p-Akt (Ser 473)蛋白的表达明显下调,而总Akt的表达没有显著变化。同时,Bax的表达增加,而Bcl-2的表达下降,导致Bax/Bcl-2的表达比率明显上升,下游cleaved PARP的表达增加。6.IGF-1能通过上调PI3K和p-Akt蛋白的表达而活化PI3K/Akt信号通路,而辛伐他汀与之作用相反,起到了类似于LY294002的通路抑制作用,其下游的Bax/Bcl-2的表达比率也发生了对应的变化。IGF-1与辛伐他汀共同作用MNNG/HOS细胞后辛伐他汀对PI3K/Akt信号通路的抑制作用被逆转,证明辛伐他汀诱导凋亡的主要分子机制涉及PI3K/Akt信号通路的失活。7.成功利用裸鼠建立骨肉瘤细胞的动物模型。辛伐他汀对荷瘤裸鼠的体重及日常活动没有明显影响,具有较好的耐受性,辛伐他汀组的移植瘤体积要明显小于对照组肿瘤体积,证实了辛伐他汀抗骨肉瘤作用的有效性。结论1.辛伐他汀能够有效的抑制体外骨肉瘤细胞系的增殖并明显抑制动物模型中骨肉瘤的生长。2. 辛伐他汀抑制骨肉瘤细胞迁移和侵袭,可能与下调MMP-2和-9的表达有关。3.辛伐他汀通过下调细胞周期调节蛋白cyclin D1, CDK2和CDK4表达而上调p21和p27蛋白表达诱导G0/G1周期阻滞。4.辛伐他汀诱导骨肉瘤细胞凋亡,可能与PI3K/Akt信号通路失活有关。
[Abstract]:Research background osteosarcoma is the most common primary malignant bone tumor originating in the skeletal system, mainly in children and adolescents. Lung metastasis is the most common early metastasis site of osteosarcoma and is also an important cause of death. The standard treatment of osteosarcoma at present is the standard treatment for osteosarcoma. Neoadjuvant chemotherapy, surgical resection, and postoperative adjuvant chemotherapy. However, even with extensive surgical excision and high intensity adjuvant chemotherapy, tumor recurrence or metastasis will occur in approximately 35-55% of first diagnosed tumor patients. The presence of chemotherapy resistance and the side effects of chemotherapy further affect the treatment of osteosarcoma Therefore, it is necessary to continue to develop new therapeutic drugs to further improve the effect of osteosarcoma treatment and improve the prognosis. The allicin is a natural organic sulfide (organosulfur compounds, OSCs) extracted from the bulbs of garlic, a mixture of many volatile allyl sulfides, the main components including the diallyl sulphide vulcanization. Substances (diallyl sulfide, DAS), diallyl two sulfides (diallyl disulfide, DADS), diallyl three sulfides (diallyl trisulfide, DATS) and allicene (Ajoene). In recent years, epidemiological studies have found that the intake of garlic is negatively related to the occurrence of some tumors, and many in vivo and in vitro experiments have also confirmed that allicin is obvious. Among the various active ingredients of allicin, DATS shows a potential anti-cancer drug that is stronger than several other components. Previous studies have shown that DATS has an obvious antitumor effect on gastric cancer, colon cancer, lung cancer, breast cancer, and prostate cancer. However, the role and mechanism of DATS on osteosarcoma In this study, two cells of osteosarcoma cell line MG63 and MNNG/HOS were used as research objects to study the effect of DATS on two kinds of cells and the possible molecular mechanisms involved, in order to provide a theoretical basis for the use of DATS for the treatment of osteosarcoma. Objective 1. to observe the effect of DATS on osteosarcoma cells by.2. to explore the inhibitory bone of the bone. The possible mechanism of action of sarcoma growth. 1. the use of different concentrations of DATS to treat osteosarcoma cells MG63 and MNNG/HOS 24h, 48h, 72h, CCK-8 method was used to detect the cell activity of osteosarcoma cells, and the effect of DATS on the proliferation of osteosarcoma cells was analyzed. The application of DATS incubating MG63 and MNNG/HOS cells was observed under the inverted microscope. Morphological changes of cells, the morphological changes of osteosarcoma cells were observed by DATS..3. cell cycle detection kit was used to detect the cell cycle distribution changes after DATS treatment of osteosarcoma cells MG63 and MNNG/HOS. The effect of DATS on the cell cycle distribution of osteosarcoma cells was observed. Western Blot was used to detect the cell cycle related proteins cyclin D1, p21, p27. Expression and analysis of the possible molecular mechanism of DATS induced osteosarcoma cell G0/G1 cycle arrest.4. application cell apoptosis detection kit to detect the cell apoptosis ratio after DATS treatment of osteosarcoma cells MG63 and MNNG/HOS, observe the effect of DATS on osteosarcoma cell apoptosis,.5. application fluorescence probe DCFH-DA through fluorescence microscopy and flow cytometry Detection of DATS induced MG63 and MNNG/HOS intracellular reactive oxygen (ROS) change level.6. application fluorescence probe JC-1 to detect the mitochondrial membrane potential changes of MG63 and MNNG/HOS after DATS treatment.7. DATS treatment of MG63 and purified cells. Expression of osoho-Akt and the expression of downstream molecules Bad, Bax, Bcl-2, Bcl-XL, to detect the changes in the expression of cytochrome c, caspase-9, Caspase-3, cleaved PARP protein in the mitochondrial apoptosis pathway. The possible molecular mechanism of apoptosis of osteosarcoma induced osteosarcoma cells was analyzed. Experimental results 1. The increase of concentration and treatment time increased the inhibitory effect of.DATS on the activity of MG63 cells. After.2. DATS processing cell 48h, the osteosarcoma cells had obvious morphological changes..3. DATS induced MG63 and MNNG/HOS cells to induce MG63 and MNNG/HOS cell cycle arrest, and antioxidant NAC could reverse DATS induced periodic block to treat bone meat. After the tumor cells, the expression of cyclin D1 protein was downregulated, while the expression of p21 and p27 protein was obviously up regulated by.4. DATS, which could induce apoptotic.NAC pretreated cells in osteosarcoma cells in a dose and time dependent manner, and could reverse DATS induced apoptotic.5. DATS action MG63 and MNNG/HOS. Prolonging the action time and obviously increasing the intracellular ROS level of osteosarcoma cells.NAC completely eliminated the increase of ROS and the effect of.6. DATS on osteosarcoma cells, the mitochondrial membrane potential decreased significantly, NAC against the mitochondrial membrane potential damage effect of DATS,.7. DATS after.7. DATS, PI3Kp11o beta, PI3Kp85a, Akt, and protein tables The expression of Bad and Bax was up-regulated, while the expression of Bad and Bax was up, while the expression of Bcl-2 and Bcl-XL was down, the level of caspase-9 and caspase-3 activated in the cells and the expression of cleaved PARP protein increased obviously. I3K/Akt signaling pathway played a similar role in the PI3K/Akt signaling pathway inhibitor LY294002, while LY294002 and DATS played a significant synergistic inhibitory effect when.NAC completely reversed the DATS down regulation of the PI3K/Akt pathway protein, indicating that the DATS induced osteosarcoma cell MG63 and MNNG/HOS apoptosis is dependent on ROS increases. Conclusion 1. DATS can inhibit the growth of osteosarcoma cells obviously..2. DATS can induce ROS production and downregulation of cyclin D1, up regulation of p21 and p27 protein expression and induce G0/G1 cycle block of osteosarcoma cells,.3. DATS is induced to lead to the decrease of mitochondrial membrane potential in osteosarcoma cells. DATS induced osteosarcoma cell apoptosis by increasing ROS, its molecular mechanism may be related to the downregulation of ROS dependent PI3K/Akt signaling pathway and the activation of mitochondrial apoptosis pathway. Part second the role and mechanism of simvastatin on osteosarcoma cells Malignant bone tumors are one of the main causes of death in juvenile tumors. Even with extensive surgical resection and neoadjuvant chemotherapy, the treatment effect of osteosarcoma is still poor and the 5 year survival rate has not been significantly improved over the past twenty years. Therefore, the effective drugs for the study or development of new osteosarcoma are forced to The statin is a competitive inhibitor of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and the HMG-CoA reductase is a speed limiting enzyme in the endogenous cholesterol synthesis in the human body. Statins can effectively reduce intracellular cholesterol by inhibiting the activity of HMG-CoA reductase. In recent years, the antitumor effects of statins have gained more and more attention and have been shown to inhibit the growth of various tumors and promote their apoptosis. In many statins, simvastatin has shown more in vivo, in vitro and in animal experiments. Previous in vitro studies have proved that simvastatin can effectively inhibit the growth of ovarian, cholangiocarcinoma, kidney, prostate and liver cancer. The main anti-tumor mechanism is to induce apoptosis and cycle arrest. However, the effect of simvastatin on osteosarcoma is less, and its anti osteosarcoma effect and the effect of simvastatin are less. This study will detect the role of simvastatin on osteosarcoma by a series of methods and explore the possible molecular mechanism to provide a theoretical basis for the possible prospect of simvastatin in the treatment of osteosarcoma. 1. the effect of simvastatin on osteosarcoma cells was observed by.2. exploration of simvastatin for the inhibition of osteosarcoma. Long possible signaling pathways and molecular mechanisms. Method 1. the effects of different concentrations of simvastatin on osteosarcoma cells MNNG/HOS 24h, 48h, 72h were used to detect the cell activity of osteosarcoma cells by CCK-8, and the effects of simvastatin on the proliferation of osteosarcoma cells were analyzed by.2. application cell scratch test (callus test) to detect simvastatin treatment of bone and meat Cell migration distance after tumor cell MNNG/HOS, the effect of simvastatin on osteosarcoma cell migration ability was observed. The effect of simvastatin on the invasion ability of osteosarcoma cells was detected by the Transwell chamber with Matrigel matrix glue at the bottom of.3. application. Western Blot was used to detect the matrix metalloproteinase (MMPs) MMP-2, the expression of MMP-9 protein and the analysis of symplectic protein. The possible molecular mechanism of lovastatin inhibition of osteosarcoma cell migration and invasion.4. application cell cycle detection kit to detect the cell cycle distribution changes after simvastatin treatment of osteosarcoma cell MNNG/HOS, and observe the effect of simvastatin on the cell cycle distribution of osteosarcoma cells. Western Blot was used to detect cell cycle related protein cyclin D1, Expression of CDK2, CDK4, p21, p27, the possible molecular mechanism of GO/G1 cycle arrest induced by simvastatin in osteosarcoma cells.5. application cell apoptosis detection kit to detect the effect of simvastatin on the apoptosis of osteosarcoma cells. Western Blot was used to detect PI3K, Akt, p-Akt, and downstream. Changes in white expression. Analysis of the possible molecular mechanism of simvastatin induced osteosarcoma cell apoptosis.6. use PI3K/Akt signaling pathway activator IGF-1 and specific inhibitor LY294002 and different concentrations of simvastatin, respectively, to extract total protein, and detect the expression of PI3K, Akt, p-Akt, and Bax, Bcl-2, cleave downstream of the MNNG/HOS cells. Changes in the expression of D PARP protein, analysis of the role of PI3K/Akt signaling pathway in the apoptosis of osteosarcoma induced by simvastatin.7. to establish an animal model of osteosarcoma MNNG/HOS cells, give intraperitoneal injection of simvastatin or physiological saline, weigh the body weight of nude mice regularly and measure the volume of the transplanted tumor. Observe the bone meat of simvastatin in animal model Experimental results 1. simvastatin inhibits the proliferation of osteosarcoma cell MNNG/HOS in time and concentration dependent manner,.2. simvastatin inhibits the migration of MNNG/HOS in osteosarcoma cells, and dose dependent.3. simvastatin inhibits the invasion of osteosarcoma cells MNNG/HOS, and is dose-dependent. Meanwhile, MM in MNNG/HOS cells The expression of P-2 and MMP-9 protein decreased obviously. The molecular mechanism of inhibition of osteosarcoma cell invasion by simvastatin may be related to the down-regulation of MMP-2 and MMP-9 protein expression related to.4. simvastatin induced MNNG/HOS cell G0/G1 cycle block.Western Blot results, and the expression of cyclin D1, CDK2, protein expression in cell is obviously down. The expression of white was obviously up-regulated, which explained the possible molecular mechanism of G0/G1 cycle block in osteosarcoma induced by simvastatin. After the action of.5. simvastatin, the apoptosis ratio of MNNG/HOS cells increased significantly and the dose positive correlation.Western blot results showed that the expression of PI3K and p-Akt (Ser 473) protein was obvious after the use of simvastatin. At the same time, the expression of total Akt did not change significantly. At the same time, the expression of Bax increased, while the expression of Bcl-2 decreased, resulting in a significant increase in the expression ratio of Bax/Bcl-2. The expression of.6.IGF-1 in the downstream cleaved PARP can activate the PI3K/Akt signaling pathway by up regulation of the expression of PI3K and p-Akt protein, while simvastatin is the opposite of the action of simvastatin. LY294002 pathway inhibition, the downstream Bax/Bcl-2 expression ratio also has a corresponding change in the inhibitory effect of simvastatin on the PI3K/Akt signaling pathway after the co action of simvastatin and simvastatin. It is proved that the main molecular mechanism of simvastatin induced apoptosis involves the inactivation of the PI3K/Akt signaling pathway in.7. formation. Animal models of osteosarcoma cells were established in nude mice. Simvastatin did not show significant weight and daily activities in nude mice bearing tumor.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R738
[Abstract]:Research background osteosarcoma is the most common primary malignant bone tumor originating in the skeletal system, mainly in children and adolescents. Lung metastasis is the most common early metastasis site of osteosarcoma and is also an important cause of death. The standard treatment of osteosarcoma at present is the standard treatment for osteosarcoma. Neoadjuvant chemotherapy, surgical resection, and postoperative adjuvant chemotherapy. However, even with extensive surgical excision and high intensity adjuvant chemotherapy, tumor recurrence or metastasis will occur in approximately 35-55% of first diagnosed tumor patients. The presence of chemotherapy resistance and the side effects of chemotherapy further affect the treatment of osteosarcoma Therefore, it is necessary to continue to develop new therapeutic drugs to further improve the effect of osteosarcoma treatment and improve the prognosis. The allicin is a natural organic sulfide (organosulfur compounds, OSCs) extracted from the bulbs of garlic, a mixture of many volatile allyl sulfides, the main components including the diallyl sulphide vulcanization. Substances (diallyl sulfide, DAS), diallyl two sulfides (diallyl disulfide, DADS), diallyl three sulfides (diallyl trisulfide, DATS) and allicene (Ajoene). In recent years, epidemiological studies have found that the intake of garlic is negatively related to the occurrence of some tumors, and many in vivo and in vitro experiments have also confirmed that allicin is obvious. Among the various active ingredients of allicin, DATS shows a potential anti-cancer drug that is stronger than several other components. Previous studies have shown that DATS has an obvious antitumor effect on gastric cancer, colon cancer, lung cancer, breast cancer, and prostate cancer. However, the role and mechanism of DATS on osteosarcoma In this study, two cells of osteosarcoma cell line MG63 and MNNG/HOS were used as research objects to study the effect of DATS on two kinds of cells and the possible molecular mechanisms involved, in order to provide a theoretical basis for the use of DATS for the treatment of osteosarcoma. Objective 1. to observe the effect of DATS on osteosarcoma cells by.2. to explore the inhibitory bone of the bone. The possible mechanism of action of sarcoma growth. 1. the use of different concentrations of DATS to treat osteosarcoma cells MG63 and MNNG/HOS 24h, 48h, 72h, CCK-8 method was used to detect the cell activity of osteosarcoma cells, and the effect of DATS on the proliferation of osteosarcoma cells was analyzed. The application of DATS incubating MG63 and MNNG/HOS cells was observed under the inverted microscope. Morphological changes of cells, the morphological changes of osteosarcoma cells were observed by DATS..3. cell cycle detection kit was used to detect the cell cycle distribution changes after DATS treatment of osteosarcoma cells MG63 and MNNG/HOS. The effect of DATS on the cell cycle distribution of osteosarcoma cells was observed. Western Blot was used to detect the cell cycle related proteins cyclin D1, p21, p27. Expression and analysis of the possible molecular mechanism of DATS induced osteosarcoma cell G0/G1 cycle arrest.4. application cell apoptosis detection kit to detect the cell apoptosis ratio after DATS treatment of osteosarcoma cells MG63 and MNNG/HOS, observe the effect of DATS on osteosarcoma cell apoptosis,.5. application fluorescence probe DCFH-DA through fluorescence microscopy and flow cytometry Detection of DATS induced MG63 and MNNG/HOS intracellular reactive oxygen (ROS) change level.6. application fluorescence probe JC-1 to detect the mitochondrial membrane potential changes of MG63 and MNNG/HOS after DATS treatment.7. DATS treatment of MG63 and purified cells. Expression of osoho-Akt and the expression of downstream molecules Bad, Bax, Bcl-2, Bcl-XL, to detect the changes in the expression of cytochrome c, caspase-9, Caspase-3, cleaved PARP protein in the mitochondrial apoptosis pathway. The possible molecular mechanism of apoptosis of osteosarcoma induced osteosarcoma cells was analyzed. Experimental results 1. The increase of concentration and treatment time increased the inhibitory effect of.DATS on the activity of MG63 cells. After.2. DATS processing cell 48h, the osteosarcoma cells had obvious morphological changes..3. DATS induced MG63 and MNNG/HOS cells to induce MG63 and MNNG/HOS cell cycle arrest, and antioxidant NAC could reverse DATS induced periodic block to treat bone meat. After the tumor cells, the expression of cyclin D1 protein was downregulated, while the expression of p21 and p27 protein was obviously up regulated by.4. DATS, which could induce apoptotic.NAC pretreated cells in osteosarcoma cells in a dose and time dependent manner, and could reverse DATS induced apoptotic.5. DATS action MG63 and MNNG/HOS. Prolonging the action time and obviously increasing the intracellular ROS level of osteosarcoma cells.NAC completely eliminated the increase of ROS and the effect of.6. DATS on osteosarcoma cells, the mitochondrial membrane potential decreased significantly, NAC against the mitochondrial membrane potential damage effect of DATS,.7. DATS after.7. DATS, PI3Kp11o beta, PI3Kp85a, Akt, and protein tables The expression of Bad and Bax was up-regulated, while the expression of Bad and Bax was up, while the expression of Bcl-2 and Bcl-XL was down, the level of caspase-9 and caspase-3 activated in the cells and the expression of cleaved PARP protein increased obviously. I3K/Akt signaling pathway played a similar role in the PI3K/Akt signaling pathway inhibitor LY294002, while LY294002 and DATS played a significant synergistic inhibitory effect when.NAC completely reversed the DATS down regulation of the PI3K/Akt pathway protein, indicating that the DATS induced osteosarcoma cell MG63 and MNNG/HOS apoptosis is dependent on ROS increases. Conclusion 1. DATS can inhibit the growth of osteosarcoma cells obviously..2. DATS can induce ROS production and downregulation of cyclin D1, up regulation of p21 and p27 protein expression and induce G0/G1 cycle block of osteosarcoma cells,.3. DATS is induced to lead to the decrease of mitochondrial membrane potential in osteosarcoma cells. DATS induced osteosarcoma cell apoptosis by increasing ROS, its molecular mechanism may be related to the downregulation of ROS dependent PI3K/Akt signaling pathway and the activation of mitochondrial apoptosis pathway. Part second the role and mechanism of simvastatin on osteosarcoma cells Malignant bone tumors are one of the main causes of death in juvenile tumors. Even with extensive surgical resection and neoadjuvant chemotherapy, the treatment effect of osteosarcoma is still poor and the 5 year survival rate has not been significantly improved over the past twenty years. Therefore, the effective drugs for the study or development of new osteosarcoma are forced to The statin is a competitive inhibitor of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and the HMG-CoA reductase is a speed limiting enzyme in the endogenous cholesterol synthesis in the human body. Statins can effectively reduce intracellular cholesterol by inhibiting the activity of HMG-CoA reductase. In recent years, the antitumor effects of statins have gained more and more attention and have been shown to inhibit the growth of various tumors and promote their apoptosis. In many statins, simvastatin has shown more in vivo, in vitro and in animal experiments. Previous in vitro studies have proved that simvastatin can effectively inhibit the growth of ovarian, cholangiocarcinoma, kidney, prostate and liver cancer. The main anti-tumor mechanism is to induce apoptosis and cycle arrest. However, the effect of simvastatin on osteosarcoma is less, and its anti osteosarcoma effect and the effect of simvastatin are less. This study will detect the role of simvastatin on osteosarcoma by a series of methods and explore the possible molecular mechanism to provide a theoretical basis for the possible prospect of simvastatin in the treatment of osteosarcoma. 1. the effect of simvastatin on osteosarcoma cells was observed by.2. exploration of simvastatin for the inhibition of osteosarcoma. Long possible signaling pathways and molecular mechanisms. Method 1. the effects of different concentrations of simvastatin on osteosarcoma cells MNNG/HOS 24h, 48h, 72h were used to detect the cell activity of osteosarcoma cells by CCK-8, and the effects of simvastatin on the proliferation of osteosarcoma cells were analyzed by.2. application cell scratch test (callus test) to detect simvastatin treatment of bone and meat Cell migration distance after tumor cell MNNG/HOS, the effect of simvastatin on osteosarcoma cell migration ability was observed. The effect of simvastatin on the invasion ability of osteosarcoma cells was detected by the Transwell chamber with Matrigel matrix glue at the bottom of.3. application. Western Blot was used to detect the matrix metalloproteinase (MMPs) MMP-2, the expression of MMP-9 protein and the analysis of symplectic protein. The possible molecular mechanism of lovastatin inhibition of osteosarcoma cell migration and invasion.4. application cell cycle detection kit to detect the cell cycle distribution changes after simvastatin treatment of osteosarcoma cell MNNG/HOS, and observe the effect of simvastatin on the cell cycle distribution of osteosarcoma cells. Western Blot was used to detect cell cycle related protein cyclin D1, Expression of CDK2, CDK4, p21, p27, the possible molecular mechanism of GO/G1 cycle arrest induced by simvastatin in osteosarcoma cells.5. application cell apoptosis detection kit to detect the effect of simvastatin on the apoptosis of osteosarcoma cells. Western Blot was used to detect PI3K, Akt, p-Akt, and downstream. Changes in white expression. Analysis of the possible molecular mechanism of simvastatin induced osteosarcoma cell apoptosis.6. use PI3K/Akt signaling pathway activator IGF-1 and specific inhibitor LY294002 and different concentrations of simvastatin, respectively, to extract total protein, and detect the expression of PI3K, Akt, p-Akt, and Bax, Bcl-2, cleave downstream of the MNNG/HOS cells. Changes in the expression of D PARP protein, analysis of the role of PI3K/Akt signaling pathway in the apoptosis of osteosarcoma induced by simvastatin.7. to establish an animal model of osteosarcoma MNNG/HOS cells, give intraperitoneal injection of simvastatin or physiological saline, weigh the body weight of nude mice regularly and measure the volume of the transplanted tumor. Observe the bone meat of simvastatin in animal model Experimental results 1. simvastatin inhibits the proliferation of osteosarcoma cell MNNG/HOS in time and concentration dependent manner,.2. simvastatin inhibits the migration of MNNG/HOS in osteosarcoma cells, and dose dependent.3. simvastatin inhibits the invasion of osteosarcoma cells MNNG/HOS, and is dose-dependent. Meanwhile, MM in MNNG/HOS cells The expression of P-2 and MMP-9 protein decreased obviously. The molecular mechanism of inhibition of osteosarcoma cell invasion by simvastatin may be related to the down-regulation of MMP-2 and MMP-9 protein expression related to.4. simvastatin induced MNNG/HOS cell G0/G1 cycle block.Western Blot results, and the expression of cyclin D1, CDK2, protein expression in cell is obviously down. The expression of white was obviously up-regulated, which explained the possible molecular mechanism of G0/G1 cycle block in osteosarcoma induced by simvastatin. After the action of.5. simvastatin, the apoptosis ratio of MNNG/HOS cells increased significantly and the dose positive correlation.Western blot results showed that the expression of PI3K and p-Akt (Ser 473) protein was obvious after the use of simvastatin. At the same time, the expression of total Akt did not change significantly. At the same time, the expression of Bax increased, while the expression of Bcl-2 decreased, resulting in a significant increase in the expression ratio of Bax/Bcl-2. The expression of.6.IGF-1 in the downstream cleaved PARP can activate the PI3K/Akt signaling pathway by up regulation of the expression of PI3K and p-Akt protein, while simvastatin is the opposite of the action of simvastatin. LY294002 pathway inhibition, the downstream Bax/Bcl-2 expression ratio also has a corresponding change in the inhibitory effect of simvastatin on the PI3K/Akt signaling pathway after the co action of simvastatin and simvastatin. It is proved that the main molecular mechanism of simvastatin induced apoptosis involves the inactivation of the PI3K/Akt signaling pathway in.7. formation. Animal models of osteosarcoma cells were established in nude mice. Simvastatin did not show significant weight and daily activities in nude mice bearing tumor.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R738
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