大蒜素对人骨肉瘤细胞株Saos-2细胞作用效果的蛋白质组学研究
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摘要
研究背景
骨肉瘤是常见的原发性骨恶性肿瘤,好发于青少年,由于肿瘤呈浸润性生长,使各种治疗手段均难以达到根治程度。1970年以前标准治疗是截肢术,但5年生存率仅约为10%。近30年来,随着新辅助化疗的广泛应用,骨肉瘤的5年生存率已达60%。我国目前未行术前化疗者复发率在30%左右,应用术前化疗后60%～70%患者得以保肢。一些研究还表明,新辅助化疗联合保肢手术治疗后的患者,其5年生存率可达70%。而我们的临床研究也发现,应用新辅助化疗和保肢手术治疗后(随访时间平均为47个月),患者的无瘤生存率为54.3%。因此,尽管化疗在骨肉瘤治疗中具有举足轻重的地位并取得了可喜的成就,但是骨肉瘤化疗总体有效率仍徘徊在60%左右。制约其疗效的主要因素在于两个方面,一是高剂量强度化疗药物所导致的严重毒副作用,如骨髓抑制、胃肠道反应、心脏毒性、肝脏毒性、肾脏及膀胱毒性、神经系统毒性、过敏反应及皮肤毒性、肺部不良反应等;二是肿瘤细胞原发或继发的耐药问题。因此,开发和选用毒副作用低、骨肉瘤细胞没有耐药性或耐药性较低的药物成为当前骨肉瘤化疗新的突破点。
大蒜素(diallyl trisulfide,DATS,化学名二烯丙基三硫化物),是从粉碎新鲜大蒜中提取的一种有机硫化合物,具有良好的抗肿瘤作用。许多研究结果表明,大蒜素抗肿瘤的作用机理主要是两个方面:停滞细胞周期和/或诱导细胞凋亡。通过对细胞周期依赖性激酶(Cdks)和细胞周期调节蛋白(cyclins)的调节来阻止细胞有丝分裂、停滞细胞周期;通过线粒体信号途径(主要是影响Bcl-2家族)、钙离子信号途径、氧化应激途径来激活caspase活性,最终诱导细胞凋亡,从而达到抑制细胞增殖的目的。但是就目前的研究来看,针对大蒜素的应用似乎局限于了腺上皮来源的癌,没有相关的资料证明大蒜素抑制肿瘤生长有部位或组织来源的选择性。因此,本课题以人骨肉瘤细胞株Saos-2细胞为研究对象,进行体外实验,以研究大蒜素对人骨肉瘤细胞株的作用效果,并探讨其抑制人骨肉瘤细胞增殖的作用机理。本研究首次将大蒜素应用到治疗骨肉瘤的实验研究当中,并用目前的热点研究手段—蛋白质组学技术来探讨大蒜素对骨肉瘤细胞的作用机理,不仅拓展了大蒜素抗肿瘤的作用范围,并可为药物治疗骨肉瘤提供新的研究方向。本研究大体可分为两个部分:
一、大蒜素对人骨肉瘤细胞株saos-2细胞增殖抑制作用的实验研究
目的
尽管目前许多的研究已经表明,大蒜素可通过停滞细胞周期和/或诱导细胞凋亡达到抑制细胞增殖的目的。但是就目前的研究来看,针对大蒜素的应用似乎局限于了腺上皮来源的癌,没有相关的资料证明大蒜素抑制肿瘤生长有部位或组织来源的选择性。
本研究的目的就是以人骨肉瘤细胞株Saos-2细胞为研究对象,应用各种不同的方法检测大蒜素作用后,Saos-2细胞形态及细胞周期、细胞凋亡等细胞生物学特征的变化,并通过这些变化来分析大蒜素对人骨肉瘤细胞株Saos-2细胞增殖的抑制效果。
方法
1倒置显微镜观察不同浓度的大蒜素作用不同时间段后,Saos-2细胞形态变化。
2不同浓度的大蒜素作用于Saos-2细胞不同时间段后,MTT法检测大蒜素对细胞增值的抑制效果。
3不同浓度的大蒜素作用于Saos-2细胞不同时间段后,应用流式细胞仪检测Saos-2细胞周期。
4不同浓度的大蒜素作用于Saos-2细胞不同时间段后,应用流式细胞仪检测Saos-2细胞凋亡。
结果
1不同浓度的大蒜素作用不同时间段后,Saos-2细胞的形态变化
将25μM,50μM,100μM大蒜素加入到人骨肉瘤细胞株Saos-2细胞的培养基中,分别处理24、48、72和96小时后,倒置显微镜观察细胞形态变化,结果发现,对照组细胞排列规则,连接紧密,大小均匀,细胞形态及胞膜界限清晰,核呈圆形并位于细胞中央。而大蒜素组细胞之间连接减少,形态不规则,内部颗粒增多,部分细胞变圆、皱缩,胞膜外突,并出现脱落死亡细胞。随着浓度的增加和时间的延长,上述改变更加明显。
2大蒜素对Saos-2细胞增殖的影响
25μM,50μM,100μM大蒜素分别处理24、48、72和96小时后,MTT法检测细胞增殖,结果发现,大蒜素对Saos-2细胞生长有明显的抑制作用,与空白组对比有显著性差异,并且有浓度和时间的依赖性。
3大蒜素对Saos-2细胞周期的影响
25μM,50μM,100μM大蒜素分别处理24、48、72和96小时后,流式细胞仪检测细胞周期发现,大蒜素可使Saos-2细胞停滞在G0/G1期,并且随着浓度和时间的增加,停滞在G0/G1期的细胞比例也随之增加。
4大蒜素对Saos-2细胞凋亡的影响
25μM,50μM,100μM大蒜素分别处理24、48、72和96小时后,流式细胞仪检测细胞凋亡发现,大蒜素可诱导细胞凋亡,并且其作用效果有一定的浓度和时间相关性。
二、大蒜素抑制人骨肉瘤细胞株Saos-2细胞增殖的分子机制研究
目的
通过大蒜素对人骨肉瘤细胞株Saos-2细胞增殖抑制作用的实验研究,我们可以得出这样的结论,大蒜素可通过停滞细胞周期进程和/或诱导细胞凋亡以达到其抑制Saos-2细胞增殖的作用效果。
本研究的目的就是运用蛋白质组学技术,从蛋白水平检测、分析人骨肉瘤细胞周期和/或细胞凋亡相关蛋白的变化,探讨大蒜素抑制人骨肉瘤细胞增殖的分子作用机制。
方法
1 50μM大蒜素作用于Saos-2细胞48小时后,提取细胞总蛋白,双向凝胶电泳技术(two-dimensional electrophoresis,2-DE)初步分析大蒜素作用前后的蛋白质表达变化。
2切取表达变化显著的蛋白质点转入质谱分析,得出新的或已知的蛋白质。
结果
1双向凝胶电泳
从Saos-2细胞中提取的蛋白在2-DE上得到很好的解析。在2-DE图像上,50μM大蒜素组和对照组Saos-2细胞总蛋白在相同的模式中电泳。2-DE图像扫描后,ImageMaster 2D Platinum进行分析,对照组和实验组得到的总的点数分别为316±6(n=3)和310±3(n=3)。点容积3倍的变化,设定为显著变化的界限。结果共有36个点被认为是对大蒜素敏感的,其中22个点是下调的,14个点是上调的。
2质谱分析
切取36个对大蒜素敏感的点,胰蛋白酶消化后,转入基质辅助激光解析/电离飞行时间质谱分析(MALDI-TOF MS)。根据质谱仪所得的数据,27个点与蛋白质匹配,其中包括下调18个,上调9个。这些蛋白参与到许多细胞生物学进程中,比如细胞生长周期和凋亡、细胞构架和基础代谢、以及蛋白合成和分解等。
结论
本课题首次将大蒜素应用到治疗骨肉瘤的实验研究当中,以人骨肉瘤细胞株Saos-2细胞为研究对象,进行体外实验,倒置显微镜观察细胞形态变化,MTT法检测大蒜素对细胞增值的抑制效果,结果发现大蒜素可抑制Saos-2细胞的增殖,并有浓度和时间的依赖性。流式细胞仪检测发现大蒜素可使Saos-2细胞停滞在G0/G1期,并且可诱导细胞凋亡。进一步应用双向电泳和质谱分析技术研究发现,大蒜素可使Saos-2细胞的一些蛋白表达发生显著变化,而这些蛋白在许多细胞生物学进程中发挥重要的作用。
本研究表明,大蒜素可通过停滞细胞周期进程和/或诱导细胞凋亡以达到其抑制人骨肉瘤细胞株Saos-2细胞增殖的作用效果,这不仅拓宽了大蒜素抗肿瘤的应用范围,也有望成为药物治疗骨肉瘤新的研究方向。而蛋白质组学的研究结果不仅为我们提示了大蒜素抗肿瘤的分子水平机制,并且可为我们今后研究骨肉瘤提供新的作用靶点。
Background
Osteosarcoma is a primary malignant tumor of the skeleton, which frequently occurs in adolescent. Various therapeutic tools failed in radical cure, due to the character of infiltrating growth in osteosarcoma. Before 1970, all patients with osteosarcoma were treated by amputation but the 5-year survival rate was under 10% and almost all patients died within a year from diagnosis. Dramatic therapeutic improvement achieved in the last 30 years is a result of development of aggressive and efficient combination chemotherapy regimens. Thus, modern treatment programmes are typically multimodal, with surgery combined with both pre- and postoperative chemotherapy (neoadjuvant chemotherapy). Today, for localised osteosarcoma at onset treated with neoadjuvant chemotherapy associated with surgery, the percentage of patients cured varies between 60% and 70%. Several studies had indicated that the five-year survival rate of patients with osteosarcoma treated with neoadjuvant chemotherapy and limb salvage surgery can be more than 70%. However, in our clinical study, the disease free survival rate was 54.5%( the mean follow-up time was 47 months ). Although neoadjuvant chemotherapy is effective in improving patient survival, the frequent acquisition of drugresistant phenotypes and the occurrence of adverse reaction, such as myelosuppression, hepatotoxicity, toxicity of kidney, toxicity of heart, toxicity of nervous system, gastrointestinal reaction, are often associated with chemotherapy, which remain as serious problems. Therefore, there is a clear need for newer effective agents, which will be a therapeutic breakthrough for osteosarcoma in chemotherapeutics.
Diallyl trisulfide(DATS), one of organosulfur compounds (OSCs) generated upon processing of fresh garlic, can inhibit proliferation of cultured cancer cells. Evidences have been accumulated to indicate that DATS can block cell mitosis and arrest cell cycle through regulating cyclin-dependent kinases (Cdks) and cyclins. Three biological processes, including mitochondrial signals, calcium homeostasis and oxidative stress, are typical events that result in or from apoptosis. Several components of apoptotic pathways have been widely investigated under DATS-induction, such as upregulation of Bax, Bad or Bak, downregulation of Bcl-2, activation of caspase-3 and -9. However, it seems that the previous studies limited on cancer cells from glandular organ or epithelial tissue. According to our available information, no related documents suggested that the anticarcinogenic effect of DATS has the selectivity in tissue origin. In the present study, we make attempt to investigate the effect of DATS on human osteosarcoma cell line Saos-2 cells, and the molecular mechanisms of DATS in suppressing cell proliferation. In this study, DATS was first applied to experimental study on drug treatment of osteosarcoma. Moreover, the combination of two-dimensional electrophoresis (2-DE) with mass spectrometry and database interrogations allowed us to identify the proteins differentially expressed in Saos-2 cells following DATS treatment. Therefore, our study can not only expand the anticarcinogenic extent of DATS, but also provide a novel insight into the drug treatment of osteosarcoma.
PARTⅠEffects of diallyl trisulfide on cell proliferation in a human osteosarcoma cell line Saos-2 cells
Objective
Evidences have been accumulated to indicate that DATS has the ability to suppress cell proliferation by blocking cell cycle progression and inducing apoptosis. However, it seems that the previous studies limited on cancer cells from glandular organ or epithelial tissue. According to our available information, no related documents suggested that the anticarcinogenic effect of DATS has the selectivity in tissue origin. In the present study, we make attempt to investigate the effect of DATS on human osteosarcoma cell line Saos-2 cells. After treated with DATS, cell morphologic change, cell cycle and apoptosis were used to analyze the effect on cell proliferation.
Methods
1 After treated with DATS at desired concentration for various time intervals, cell morphologic change was observed under inverted microscope.
2 The effect of DATS on cell proliferation was detected by MTT [3-(4-5dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide] assay.
3 After treated with DATS at desired concentration for various time intervals, cell cycle phase was analyzed by flow cytometry.
4 After treated with DATS at desired concentration for various time intervals, the apoptotic rate was analyzed by flow cytometry.
Results
1 Cell morphologic changes
After treated with 25μM, 50μM or 100μM DATS for various incubation time intervals, the shape deformations, such as shrinkage of cell bodies, the intercellular gaps becoming loose, cell lyses, condensation of nuclei, were observed under inverted microscope. In addition, many cells became round, floated and necrotic. Moreover, these morphologic changes had concentration- and time-dependent phenomenon. However, as compared to the treated groups, the cell morphology in control group had no visible changes.
2 DATS inhabits the proliferation of Saos-2 cells
The antiproliferative effect of DATS Saos-2 cells, was examined through exposing them to different concentrations of DATS (25μM, 50μM or 100μM) for 24, 48, 72h and 96h, respectively. These results indicated that DATS can exert significant inhibition to Saos-2 cells proliferation in concentration- and time-dependent manner.
3 Cell cycle analysis
The cell cycle of Saos-2 cells was arrested at G0/G1 phase when the cells were treated with DATS. The more concentration of DATS used, the more Saos-2 cells were blocked at G0/G1 phase. In addition, with the treated time prolonged, the percentage of arrest at G0/G1 phase was increased.
4 Apoptosis analysis
The results indicated that DATS can induce apoptosis in Saos-2 cells. Furthermore, the effect of DATS-induced apoptosis was increased in concentration- and time-dependent manner
PARTⅡExperimental study on the molecular mechanisms of diallyl trisulfide
Objective
In PARTⅠ, we make attempt to investigate the effects of DATS on a human osteosarcoma cell line, Saos-2 cells. The results demonstrate that DATS has the ability to suppress cell proliferation of Saos-2 cells by blocking cell cycle progression and inducing apoptosis in a dose- and time-dependent manner.
In an effort to approach the molecular mechanisms of DATS on Saos-2 cells, we used 2-DE and mass spectrometry to examine the protein profiles of Saos-2 cells treated with DATS.
Methods
1 After treated with or without 50uM DATS for 48h, total proteins in Saos-2 cells were extracted. The response of protein expression in Saos-2 cells induced by DATS was analyzed by 2-DE.
2 The gel spots verified as the significant changes in spot volume were separated and transferred into matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to obtain newer or existing proteins.
Results
1 2-DE patterns of Saos-2 cells treated with or without DATS
The proteins extracted from Saos-2 cells were resolved on 2-DE. Upon 2-DE images, the proteins in Saoa-2 cells either with or without DATS treatment behaved electrophorectically in similar modes. After scanned by Amersham Imagescanner, the image analysis was conducted with ImageMaster 2D Platinum. The total spots were 316±6 (n=3) and 310±3 (n=3) in the control and DATS-treated groups, respectively. The threshold of the significant change in 2-DE spots was defined as 3-folds of change in spot volume upon comparison of average gels between the treated and control groups. A total of 36 spots were defined as DATS-sensitive spots in Saos-2 cells, including 22 downregulated spots and 14 upregulated spots. The results indicated that a total of 36 spots were defined as DATS-sensitive spots in Saos-2 cells, including 22 downregulated spots and 14 upregulated spots.
2 Identification of differentially expressed proteins by MALDI-TOF MS
The spots from the 2-DE gel were subjected to trypsin digestion and MALDI-TOF MS analysis. On the basis of the data of mass spectrometry, 36 spots matched with the proteins, in which 27 spots were ascertained as unique proteins including 18 downregulated and 9 upregulated. These proteins are involved in many biological functions, such as cell growth and apoptosis, cell differentiation and mobility, cell structure and basic metabolism, protein biosynthesis and degradation.
Conclusion
In the present study, DATS was first applied to experimental study on drug treatment of osteosarcoma. We make attempt to investigate the effect of DATS on human osteosarcoma cell line Saos-2 cells, and the molecular mechanisms of DATS in suppressing cell proliferation. Using inverted microscope and MTT assay, we found that DATS has the ability to suppress cell proliferation of Saos-2 cells in dose- and time-dependent manner. Moreover, cell cycle phase and apoptotic rate were analyzed by flow cytometry; and the results suggested that DATS can block Saos-2 cells at G0/G1 phase and induce apoptosis. The proteomic data provided novel insight into a massive response of protein expression in Saos-2 cells induced by DATS, and demonstrated that some of DATS-sensitive proteins were related with several biology pathways.
In summary, the results of the present study indicate that DATS has the ability to suppress cell proliferation of Saos-2 cells by blocking cell cycle progression and inducing apoptosis in dose- and time-dependent manner. Therefore, our study can not only expand the anticarcinogenic extent of DATS, but also provide a novel insight into the drug treatment of osteosarcoma. In addition, the DATS-sensitive proteins in our study might elucidate the anticarcinogenic effects of DATS and be applied as therapeutic targets for gene therapy of osteosarcoma.
骨肉瘤是常见的原发性骨恶性肿瘤,好发于青少年,由于肿瘤呈浸润性生长,使各种治疗手段均难以达到根治程度。1970年以前标准治疗是截肢术,但5年生存率仅约为10%。近30年来,随着新辅助化疗的广泛应用,骨肉瘤的5年生存率已达60%。我国目前未行术前化疗者复发率在30%左右,应用术前化疗后60%～70%患者得以保肢。一些研究还表明,新辅助化疗联合保肢手术治疗后的患者,其5年生存率可达70%。而我们的临床研究也发现,应用新辅助化疗和保肢手术治疗后(随访时间平均为47个月),患者的无瘤生存率为54.3%。因此,尽管化疗在骨肉瘤治疗中具有举足轻重的地位并取得了可喜的成就,但是骨肉瘤化疗总体有效率仍徘徊在60%左右。制约其疗效的主要因素在于两个方面,一是高剂量强度化疗药物所导致的严重毒副作用,如骨髓抑制、胃肠道反应、心脏毒性、肝脏毒性、肾脏及膀胱毒性、神经系统毒性、过敏反应及皮肤毒性、肺部不良反应等;二是肿瘤细胞原发或继发的耐药问题。因此,开发和选用毒副作用低、骨肉瘤细胞没有耐药性或耐药性较低的药物成为当前骨肉瘤化疗新的突破点。
大蒜素(diallyl trisulfide,DATS,化学名二烯丙基三硫化物),是从粉碎新鲜大蒜中提取的一种有机硫化合物,具有良好的抗肿瘤作用。许多研究结果表明,大蒜素抗肿瘤的作用机理主要是两个方面:停滞细胞周期和/或诱导细胞凋亡。通过对细胞周期依赖性激酶(Cdks)和细胞周期调节蛋白(cyclins)的调节来阻止细胞有丝分裂、停滞细胞周期;通过线粒体信号途径(主要是影响Bcl-2家族)、钙离子信号途径、氧化应激途径来激活caspase活性,最终诱导细胞凋亡,从而达到抑制细胞增殖的目的。但是就目前的研究来看,针对大蒜素的应用似乎局限于了腺上皮来源的癌,没有相关的资料证明大蒜素抑制肿瘤生长有部位或组织来源的选择性。因此,本课题以人骨肉瘤细胞株Saos-2细胞为研究对象,进行体外实验,以研究大蒜素对人骨肉瘤细胞株的作用效果,并探讨其抑制人骨肉瘤细胞增殖的作用机理。本研究首次将大蒜素应用到治疗骨肉瘤的实验研究当中,并用目前的热点研究手段—蛋白质组学技术来探讨大蒜素对骨肉瘤细胞的作用机理,不仅拓展了大蒜素抗肿瘤的作用范围,并可为药物治疗骨肉瘤提供新的研究方向。本研究大体可分为两个部分:
一、大蒜素对人骨肉瘤细胞株saos-2细胞增殖抑制作用的实验研究
目的
尽管目前许多的研究已经表明,大蒜素可通过停滞细胞周期和/或诱导细胞凋亡达到抑制细胞增殖的目的。但是就目前的研究来看,针对大蒜素的应用似乎局限于了腺上皮来源的癌,没有相关的资料证明大蒜素抑制肿瘤生长有部位或组织来源的选择性。
本研究的目的就是以人骨肉瘤细胞株Saos-2细胞为研究对象,应用各种不同的方法检测大蒜素作用后,Saos-2细胞形态及细胞周期、细胞凋亡等细胞生物学特征的变化,并通过这些变化来分析大蒜素对人骨肉瘤细胞株Saos-2细胞增殖的抑制效果。
方法
1倒置显微镜观察不同浓度的大蒜素作用不同时间段后,Saos-2细胞形态变化。
2不同浓度的大蒜素作用于Saos-2细胞不同时间段后,MTT法检测大蒜素对细胞增值的抑制效果。
3不同浓度的大蒜素作用于Saos-2细胞不同时间段后,应用流式细胞仪检测Saos-2细胞周期。
4不同浓度的大蒜素作用于Saos-2细胞不同时间段后,应用流式细胞仪检测Saos-2细胞凋亡。
结果
1不同浓度的大蒜素作用不同时间段后,Saos-2细胞的形态变化
将25μM,50μM,100μM大蒜素加入到人骨肉瘤细胞株Saos-2细胞的培养基中,分别处理24、48、72和96小时后,倒置显微镜观察细胞形态变化,结果发现,对照组细胞排列规则,连接紧密,大小均匀,细胞形态及胞膜界限清晰,核呈圆形并位于细胞中央。而大蒜素组细胞之间连接减少,形态不规则,内部颗粒增多,部分细胞变圆、皱缩,胞膜外突,并出现脱落死亡细胞。随着浓度的增加和时间的延长,上述改变更加明显。
2大蒜素对Saos-2细胞增殖的影响
25μM,50μM,100μM大蒜素分别处理24、48、72和96小时后,MTT法检测细胞增殖,结果发现,大蒜素对Saos-2细胞生长有明显的抑制作用,与空白组对比有显著性差异,并且有浓度和时间的依赖性。
3大蒜素对Saos-2细胞周期的影响
25μM,50μM,100μM大蒜素分别处理24、48、72和96小时后,流式细胞仪检测细胞周期发现,大蒜素可使Saos-2细胞停滞在G0/G1期,并且随着浓度和时间的增加,停滞在G0/G1期的细胞比例也随之增加。
4大蒜素对Saos-2细胞凋亡的影响
25μM,50μM,100μM大蒜素分别处理24、48、72和96小时后,流式细胞仪检测细胞凋亡发现,大蒜素可诱导细胞凋亡,并且其作用效果有一定的浓度和时间相关性。
二、大蒜素抑制人骨肉瘤细胞株Saos-2细胞增殖的分子机制研究
目的
通过大蒜素对人骨肉瘤细胞株Saos-2细胞增殖抑制作用的实验研究,我们可以得出这样的结论,大蒜素可通过停滞细胞周期进程和/或诱导细胞凋亡以达到其抑制Saos-2细胞增殖的作用效果。
本研究的目的就是运用蛋白质组学技术,从蛋白水平检测、分析人骨肉瘤细胞周期和/或细胞凋亡相关蛋白的变化,探讨大蒜素抑制人骨肉瘤细胞增殖的分子作用机制。
方法
1 50μM大蒜素作用于Saos-2细胞48小时后,提取细胞总蛋白,双向凝胶电泳技术(two-dimensional electrophoresis,2-DE)初步分析大蒜素作用前后的蛋白质表达变化。
2切取表达变化显著的蛋白质点转入质谱分析,得出新的或已知的蛋白质。
结果
1双向凝胶电泳
从Saos-2细胞中提取的蛋白在2-DE上得到很好的解析。在2-DE图像上,50μM大蒜素组和对照组Saos-2细胞总蛋白在相同的模式中电泳。2-DE图像扫描后,ImageMaster 2D Platinum进行分析,对照组和实验组得到的总的点数分别为316±6(n=3)和310±3(n=3)。点容积3倍的变化,设定为显著变化的界限。结果共有36个点被认为是对大蒜素敏感的,其中22个点是下调的,14个点是上调的。
2质谱分析
切取36个对大蒜素敏感的点,胰蛋白酶消化后,转入基质辅助激光解析/电离飞行时间质谱分析(MALDI-TOF MS)。根据质谱仪所得的数据,27个点与蛋白质匹配,其中包括下调18个,上调9个。这些蛋白参与到许多细胞生物学进程中,比如细胞生长周期和凋亡、细胞构架和基础代谢、以及蛋白合成和分解等。
结论
本课题首次将大蒜素应用到治疗骨肉瘤的实验研究当中,以人骨肉瘤细胞株Saos-2细胞为研究对象,进行体外实验,倒置显微镜观察细胞形态变化,MTT法检测大蒜素对细胞增值的抑制效果,结果发现大蒜素可抑制Saos-2细胞的增殖,并有浓度和时间的依赖性。流式细胞仪检测发现大蒜素可使Saos-2细胞停滞在G0/G1期,并且可诱导细胞凋亡。进一步应用双向电泳和质谱分析技术研究发现,大蒜素可使Saos-2细胞的一些蛋白表达发生显著变化,而这些蛋白在许多细胞生物学进程中发挥重要的作用。
本研究表明,大蒜素可通过停滞细胞周期进程和/或诱导细胞凋亡以达到其抑制人骨肉瘤细胞株Saos-2细胞增殖的作用效果,这不仅拓宽了大蒜素抗肿瘤的应用范围,也有望成为药物治疗骨肉瘤新的研究方向。而蛋白质组学的研究结果不仅为我们提示了大蒜素抗肿瘤的分子水平机制,并且可为我们今后研究骨肉瘤提供新的作用靶点。
Background
Osteosarcoma is a primary malignant tumor of the skeleton, which frequently occurs in adolescent. Various therapeutic tools failed in radical cure, due to the character of infiltrating growth in osteosarcoma. Before 1970, all patients with osteosarcoma were treated by amputation but the 5-year survival rate was under 10% and almost all patients died within a year from diagnosis. Dramatic therapeutic improvement achieved in the last 30 years is a result of development of aggressive and efficient combination chemotherapy regimens. Thus, modern treatment programmes are typically multimodal, with surgery combined with both pre- and postoperative chemotherapy (neoadjuvant chemotherapy). Today, for localised osteosarcoma at onset treated with neoadjuvant chemotherapy associated with surgery, the percentage of patients cured varies between 60% and 70%. Several studies had indicated that the five-year survival rate of patients with osteosarcoma treated with neoadjuvant chemotherapy and limb salvage surgery can be more than 70%. However, in our clinical study, the disease free survival rate was 54.5%( the mean follow-up time was 47 months ). Although neoadjuvant chemotherapy is effective in improving patient survival, the frequent acquisition of drugresistant phenotypes and the occurrence of adverse reaction, such as myelosuppression, hepatotoxicity, toxicity of kidney, toxicity of heart, toxicity of nervous system, gastrointestinal reaction, are often associated with chemotherapy, which remain as serious problems. Therefore, there is a clear need for newer effective agents, which will be a therapeutic breakthrough for osteosarcoma in chemotherapeutics.
Diallyl trisulfide(DATS), one of organosulfur compounds (OSCs) generated upon processing of fresh garlic, can inhibit proliferation of cultured cancer cells. Evidences have been accumulated to indicate that DATS can block cell mitosis and arrest cell cycle through regulating cyclin-dependent kinases (Cdks) and cyclins. Three biological processes, including mitochondrial signals, calcium homeostasis and oxidative stress, are typical events that result in or from apoptosis. Several components of apoptotic pathways have been widely investigated under DATS-induction, such as upregulation of Bax, Bad or Bak, downregulation of Bcl-2, activation of caspase-3 and -9. However, it seems that the previous studies limited on cancer cells from glandular organ or epithelial tissue. According to our available information, no related documents suggested that the anticarcinogenic effect of DATS has the selectivity in tissue origin. In the present study, we make attempt to investigate the effect of DATS on human osteosarcoma cell line Saos-2 cells, and the molecular mechanisms of DATS in suppressing cell proliferation. In this study, DATS was first applied to experimental study on drug treatment of osteosarcoma. Moreover, the combination of two-dimensional electrophoresis (2-DE) with mass spectrometry and database interrogations allowed us to identify the proteins differentially expressed in Saos-2 cells following DATS treatment. Therefore, our study can not only expand the anticarcinogenic extent of DATS, but also provide a novel insight into the drug treatment of osteosarcoma.
PARTⅠEffects of diallyl trisulfide on cell proliferation in a human osteosarcoma cell line Saos-2 cells
Objective
Evidences have been accumulated to indicate that DATS has the ability to suppress cell proliferation by blocking cell cycle progression and inducing apoptosis. However, it seems that the previous studies limited on cancer cells from glandular organ or epithelial tissue. According to our available information, no related documents suggested that the anticarcinogenic effect of DATS has the selectivity in tissue origin. In the present study, we make attempt to investigate the effect of DATS on human osteosarcoma cell line Saos-2 cells. After treated with DATS, cell morphologic change, cell cycle and apoptosis were used to analyze the effect on cell proliferation.
Methods
1 After treated with DATS at desired concentration for various time intervals, cell morphologic change was observed under inverted microscope.
2 The effect of DATS on cell proliferation was detected by MTT [3-(4-5dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide] assay.
3 After treated with DATS at desired concentration for various time intervals, cell cycle phase was analyzed by flow cytometry.
4 After treated with DATS at desired concentration for various time intervals, the apoptotic rate was analyzed by flow cytometry.
Results
1 Cell morphologic changes
After treated with 25μM, 50μM or 100μM DATS for various incubation time intervals, the shape deformations, such as shrinkage of cell bodies, the intercellular gaps becoming loose, cell lyses, condensation of nuclei, were observed under inverted microscope. In addition, many cells became round, floated and necrotic. Moreover, these morphologic changes had concentration- and time-dependent phenomenon. However, as compared to the treated groups, the cell morphology in control group had no visible changes.
2 DATS inhabits the proliferation of Saos-2 cells
The antiproliferative effect of DATS Saos-2 cells, was examined through exposing them to different concentrations of DATS (25μM, 50μM or 100μM) for 24, 48, 72h and 96h, respectively. These results indicated that DATS can exert significant inhibition to Saos-2 cells proliferation in concentration- and time-dependent manner.
3 Cell cycle analysis
The cell cycle of Saos-2 cells was arrested at G0/G1 phase when the cells were treated with DATS. The more concentration of DATS used, the more Saos-2 cells were blocked at G0/G1 phase. In addition, with the treated time prolonged, the percentage of arrest at G0/G1 phase was increased.
4 Apoptosis analysis
The results indicated that DATS can induce apoptosis in Saos-2 cells. Furthermore, the effect of DATS-induced apoptosis was increased in concentration- and time-dependent manner
PARTⅡExperimental study on the molecular mechanisms of diallyl trisulfide
Objective
In PARTⅠ, we make attempt to investigate the effects of DATS on a human osteosarcoma cell line, Saos-2 cells. The results demonstrate that DATS has the ability to suppress cell proliferation of Saos-2 cells by blocking cell cycle progression and inducing apoptosis in a dose- and time-dependent manner.
In an effort to approach the molecular mechanisms of DATS on Saos-2 cells, we used 2-DE and mass spectrometry to examine the protein profiles of Saos-2 cells treated with DATS.
Methods
1 After treated with or without 50uM DATS for 48h, total proteins in Saos-2 cells were extracted. The response of protein expression in Saos-2 cells induced by DATS was analyzed by 2-DE.
2 The gel spots verified as the significant changes in spot volume were separated and transferred into matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to obtain newer or existing proteins.
Results
1 2-DE patterns of Saos-2 cells treated with or without DATS
The proteins extracted from Saos-2 cells were resolved on 2-DE. Upon 2-DE images, the proteins in Saoa-2 cells either with or without DATS treatment behaved electrophorectically in similar modes. After scanned by Amersham Imagescanner, the image analysis was conducted with ImageMaster 2D Platinum. The total spots were 316±6 (n=3) and 310±3 (n=3) in the control and DATS-treated groups, respectively. The threshold of the significant change in 2-DE spots was defined as 3-folds of change in spot volume upon comparison of average gels between the treated and control groups. A total of 36 spots were defined as DATS-sensitive spots in Saos-2 cells, including 22 downregulated spots and 14 upregulated spots. The results indicated that a total of 36 spots were defined as DATS-sensitive spots in Saos-2 cells, including 22 downregulated spots and 14 upregulated spots.
2 Identification of differentially expressed proteins by MALDI-TOF MS
The spots from the 2-DE gel were subjected to trypsin digestion and MALDI-TOF MS analysis. On the basis of the data of mass spectrometry, 36 spots matched with the proteins, in which 27 spots were ascertained as unique proteins including 18 downregulated and 9 upregulated. These proteins are involved in many biological functions, such as cell growth and apoptosis, cell differentiation and mobility, cell structure and basic metabolism, protein biosynthesis and degradation.
Conclusion
In the present study, DATS was first applied to experimental study on drug treatment of osteosarcoma. We make attempt to investigate the effect of DATS on human osteosarcoma cell line Saos-2 cells, and the molecular mechanisms of DATS in suppressing cell proliferation. Using inverted microscope and MTT assay, we found that DATS has the ability to suppress cell proliferation of Saos-2 cells in dose- and time-dependent manner. Moreover, cell cycle phase and apoptotic rate were analyzed by flow cytometry; and the results suggested that DATS can block Saos-2 cells at G0/G1 phase and induce apoptosis. The proteomic data provided novel insight into a massive response of protein expression in Saos-2 cells induced by DATS, and demonstrated that some of DATS-sensitive proteins were related with several biology pathways.
In summary, the results of the present study indicate that DATS has the ability to suppress cell proliferation of Saos-2 cells by blocking cell cycle progression and inducing apoptosis in dose- and time-dependent manner. Therefore, our study can not only expand the anticarcinogenic extent of DATS, but also provide a novel insight into the drug treatment of osteosarcoma. In addition, the DATS-sensitive proteins in our study might elucidate the anticarcinogenic effects of DATS and be applied as therapeutic targets for gene therapy of osteosarcoma.
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