人大肠癌细胞转移相关的蛋白质组分析与鉴定
|
摘要
恶性肿瘤细胞的转移是导致患者死亡的主要原因，但迄今为止，对肿瘤转移机制的研究尚未获得突破性进展。筛选和鉴定在肿瘤转移中起重要作用的关键分子，寻找早期诊断的生物标志物和抗肿瘤转移的药物靶点，阐明肿瘤转移机制，是当前肿瘤转移的研究热点。
肿瘤转移是一个多步骤、多基因参与的复杂过程，肿瘤转移的全过程受以下几个因素的调控：1、在转移启动初期E-cadherin、Ig超家族、选择素、整合素等参与肿瘤细胞间的脱落以及肿瘤细胞与胞外基质的粘附；2、肿瘤细胞或间质细胞分泌的蛋白水解酶及其抑制剂，在降解胞外基质和血管的基底膜过程中发挥重要作用；3、在运动因子、生长因子、归巢因子等的作用下，肿瘤细胞迁移到转移靶器官；4、血管增生因子VEGF、bFGF、IL-8、PDGF等促进新生血管的形成，伴随着肿瘤细胞的增生，形成新的转移灶，完成转移过程。鉴于目前对肿瘤转移机制的研究往往针对单个或少数几个分子以及关注基因水平的改变，缺乏系统性和综合性的分析，因此在所有这些调控因素中，究竟哪些分子在肿瘤转移中发挥关键作用，仍有待阐明。近年来开展的蛋白质组学技术为揭示肿瘤转移机制带来了新的希望。
本研究以人大肠癌高、低转移细胞株为研究对象，采用比较蛋白质组技术鉴定了11个与大肠癌转移相关的候选蛋白，并探讨了HSP27在大肠癌转移中的作用及其功能，具体研究结果如下：
1、人大肠癌细胞系蛋白质双向凝胶电泳技术的建立及其优化
优化人结肠癌Lovo细胞系的蛋白质样品制备方法，建立人结肠癌Lovo细胞系的双向凝胶电泳(2-DE)图谱，为识别鉴定结肠癌发生发展相关蛋白质奠定基础。分别用磷酸盐缓冲液和等渗蔗糖溶液冲洗细胞，胰酶消化或直接刮刀法收集细胞后提取总蛋白质，利用固相pH梯度双向凝胶电泳技术进行分离，凝胶经银染显色后，用PDQuest软件分析2-DE图谱。结果以等渗蔗糖缓冲液冲洗细胞并直接刮刀法所提取Lovo细胞总蛋白质进行双向电泳，可获得分辨率高、重复性好的人结肠癌Lovo细胞系2-DE图谱。等渗蔗糖溶液有效的降低了盐离子对等电聚焦的影响，聚焦效果优于磷酸盐缓冲溶液处理组；皿上直接刮刀收集的方法避免引入胰酶及其对细胞的破坏，保证了细胞蛋白质的完整性和代表性。我们实验表明以等渗蔗糖溶液冲洗细胞结合皿上直接裂解是一种较好的细胞蛋白质双向电泳样品制备方法，在此基础上我们初步建立了分辨率较高且重复性好的人结肠癌Lovo细胞系蛋白质双向凝胶电泳图谱。
2、人大肠癌细胞株SW480和SW620的蛋白质组双向凝胶电泳分析
为了更好的理解大肠癌转移机理和寻找大肠癌预后的标志分子，我们运用双向凝胶电泳技术对人大肠癌高、低转移细胞株SW480和SW620细胞进行了差异表达双向凝胶电泳分析，数字化图像分析发现有72个蛋白质斑点在两组凝胶中有显著差异表达，在SW620细胞中表达量升高的蛋白质斑点有30个，在SW480细胞中表达量升高的蛋白质斑点有42个；定性差异表达分析(表达量相差10倍以上)发现22个蛋白质斑点仅在SW620中出现，而有27个蛋白质斑点只在SW480中检测到，结合软件分析和手工筛选，选取15个点清晰且表达水平改变明显的蛋白质点作为质谱鉴定的靶点。
3、双向凝胶电泳差异表达蛋白质的质谱鉴定和验证
以大肠癌高、低转移细胞株为研究对象，采用2-DE分离技术结合MALDI-TOF质谱鉴定了11个与大肠癌转移相关的蛋白，其中SW620细胞株表达上调的蛋白质有磷酸甘油酸变位酶1，磷脂酰乙醇胺结合蛋白和高迁移率族蛋白B-1，而热休克蛋白27，膜联蛋白Ⅰ，甲硫腺苷磷酸化酶，切丝蛋白1和表皮型脂肪酸结合蛋白在SW620中表达下调。大多数差异蛋白功能涉及肿瘤细胞生长、运动、粘附、凋亡等过程，研究结果为阐明大肠癌转移机制及寻找预测大肠癌转移的潜在标志物提供了理论依据。
为保证蛋白质组技术鉴定结果的可靠性，在蛋白和mRNA水平对候选蛋白进行了进一步验证。采用western blot和免疫细胞化学技术在蛋白质水平验证结果表明HSP27和COF1在低转移的SW480中高表达，且COF1仅在SW480中检测到，蛋白质水平的验证结果与比较蛋白质组的分析结果完全一致。此外，采用半定量RT-PCR技术对编码候选蛋白的mRNA水平检测结果表明：编码HMGB1和PBP的mRNA在高转移的SW620中高表达，而编码HSP27、ANXⅠ、COF1和E-FABP的mRNA在SW480中高表达，所验证的编码候选蛋白的mRNA水平验证结果与候选蛋白的蛋白质组分析结果一致，说明候选蛋白在大肠癌细胞株中的蛋白水平变化主要是由于基因的转录水平高低不同所致，也肯定了候选蛋白在高、低转移株间的差异表达。结果表明通过蛋白质组技术筛选到的转移相关蛋白是可靠的。
4、热休克蛋白27与大肠癌转移的相关性分析
为更好的理解大肠癌细胞的转移机理和寻找可能的肿瘤预后标记分子标志物，我们用蛋白质组学方法对两种不同转移潜能的SW620和SW480细胞株的差异表达蛋白进行了研究，发现热休克蛋白27在高转移潜能大肠癌细胞中的低表达。免疫细胞化学进一步验证和分析了该蛋白在SW620和SW480两种细胞中的差异表达和定位。68例临床大肠癌组织标本的免疫组织化学研究发现热休克蛋白27的表达率与年龄、性别、组织学分级、淋巴结转移和临床分期无相关性(x~2 test，p＞0.05)，但其过表达和大肠癌转移显著负相关(Fisher's exact test，p=0.035＜0.05)。结果表明，大肠癌细胞热休克蛋白27的过表达和肿瘤细胞的转移行为相关，可能在阻止其转移中发挥重要的作用。
综上所述，本研究应用比较蛋白质组技术对人大肠癌SW620和SW480两种不同转移潜能细胞株进行了对比研究，获得了11个差异表达蛋白质并对HSP27进行了初步验证。分析结果证实大肠癌细胞热休克蛋白27的过表达和肿瘤细胞的转移行为相关，在阻止大肠癌转移中发挥重要的作用，可能是阻止大肠癌转移的关键分子之一，研究结果将为抗大肠癌转移研究和药物的筛选提供有用的研究信息和药物分子靶标。
Widespread metastasis is a common phenomenon and the major lethal cause of cancer. So far, however, very little is known about the mechanisms underlying metastasis. Once the key factors in tumor metastasis were screened and identified, the drug targets and diagnosis markers could be obtained accordingly.Metastasis is a complex multistep malignant process. It has been widely reported that many molecules were involved in the complex process of tumor invasion and metastasis: 1) E-cadherin, immunoglobin superfamily, selectin and integrin have been suggested to take part in the detachment of tumor cells from the primary site and interaction of tumor cells with the surrounding extracellular matrix; 2) Matrix-degrading enzymes and their inhibitors secreted by tumor cells or mesenchymal cells have been indicated to be involved in degradation of extracellular matrix and vascular basement; 3) Some growth factors and movememt factors have been implicated to play role in migration of tumor cells into secondary sites; 4) Angiogenetic factors, such as VEGF, bFGF, EL-8 and PDGF, have been demonstrated to be important in neoangiogenesis and distant metastasis. At present, most of the research associated with metastasis so far has been focused on the genetic changes of related molecules or single or few proteins without systematic study. But little is known about the key factors to trigger tumorigenic cells to initiate further invasion and metastasis facing with so many regulators up to now.
On the basis of these considerations, proteomic strategy, combined with two-dimensional electrophoresis (2-DE) separation and mass spectrometry (MS) identification with advantage of high resolution, high reproducibility was used to separate and identify differentially expressed proteins between highly and lowly metastatic subpopulations. In this study we hoped to find out a series of protein cluster deeply involved in metastasis process and addressed the question whether there were new proteins to be associated with metastasis, moreover, to clarify the metastasis-associated function mediated by new candidate proteins. In the present study, 11 metastasis-associated proteins were seperated and identified by comparative proteome technique using established metastatic model, more importantly, the metastasis-associated clinical characterization mediated by HSP27 was further characterized.
1. Establishment and optimization of two-dimensional polyacrylamide gel electrophoresis for the proteome analysis of the human colorectal cancer cell line
To optimize the sample preparation methods for the proteome analysis of human colon adenocarcinoma cell line Lovo, to establish a high resolution and reproducible two-dimensional polyacrylamide gel electrophoresis (2DE) image, and to provide a base for identifying disease-associated proteins. For cells cultured on solid substrates, the cell layer should be washed with phosphate-buffered saline or an isotonic sucrose solution respectively. The cells can then be harvested by treating with typsin, or scraping after having been lysed directly. Immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis was used to separate the total proteins of the samples. After silver staining, PDQuest image analysis software was applied to analyze 2DE images. Finally, the total proteins of ceils, washed with isotonic sucrose solution and scraped after having been lysed directly, were used to perform 2DE. 2DE patterns with high resolution and reproducibility of human colon adenocarcinoma cell line Lovo were obtained. The isotonic sucrose solution is a particularly efficient way of minimizing salts that can interfere with the IEF dimension. The method of scraping after having been lysed directly avoids the use of proteolytic enzymes that can destroy the cells, and it makes the total proteins complete and representative. Our findings indicated that washing with isotonic sucrose solution combinated with scraping after having been lysed directly is a proper method for protein sample preparation of human colon adenocarcinoma cell line Lovo. The high-quality separation of 2-DE gel was obtained. The well-resolved, reproducible 2-DE profiles of human colon adenocarcinoma cell line Lovo have been primarily established.
2. Comparative proteomic analysis between SW620 and SW480 cell lines with different metastasis potential
To better understand the mechanism underlying the CRC metastasis and to search potential markers for CRC prognosis, differential proteome analysis on two CRC cell strains with high and low metastatic potentials, SW620 and SW480, was conducted using various proteomics approaches. By two-dimensional gel electrophoresis (2-DE), image analysis and mass spectrometry, the expression levels of 72 protein spots were found quantitatively changed between SW620 and SW480, among them the expression levels of 30 protein spots were increased in SW620 cells and the expression levels of 42 protein spots were decreased in SW620 cells. Qualitative analysis between SW620 and SW480 cells found that 22 protein spots were only detected in SW620 cells and 27 protein spots in SW480 cells. Combinated with software and manual screening, 15 differentially expressed protein spots between SW620 and SW480 were further identified by MALDI-TOF MS analysis.
3. Further identification and verification of candidate proteins
Two-dimensional (2-D) gel electrophoresis followed by matrix-assisted laser desorption/time of flight mass spectrometry was utilized to compare the protein expression profiles between two colorectal cancer (CRC) cell lines with high and low metastatic potentials, SW620 and SW480. 11 metastasis-associated proteins were identified successfully. Of the identified proteins, the expressions of phosphoglycerate mutase 1, phosphatidylethanolamine binding protein and high-mobility group box 1 were elevated in SW620 cells. However, heat shock protein 27, annexinⅠ, methylthioadenosine phosphorylase, cofilin-1 and epidermal fatty acid binding protein were down-regulated in SW620 cells. Most of the candidate proteins have been evidenced to be somehow associated with various aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, etc. These results provide the basis for searching for potential markers for CRC prognosis and give some clues to elucidate the mechanism of CRC metastasis.
Western blot, immunocytochemistry and semi-quantitative RT-PCR analysis were used to further verify candidate proteins in order to ensure the reliabilty of the proteome results. The western blot and immunocytochemisty results showed that HSP27 and COF1 were significantly higher in SW480, and that COF1 was only detected in lowly metastatic SW480 cells. Evidently, western blot results were consistent with proteome analysis. In addition, most of the identified candidate proteins were further confirmed by semi-quantitative RT-PCR analysis at mRNA level. Semi-quantitative RT-PCR analysis indicated that the mRNAs coding HMGB1 and PBP were up-regulated, but the mRNAs coding HSP27, ANX I, COF1 and E-FABP were down-regulated in highly metastatic SW620 subpopulation compared with lowly metastatic SW480. The verification results indicated that most of the differences of protein expression displayed by 2-DE between SW620 and SW480 was convincing.
4. Overexpression of HSP27 correlates with colorectal carcinoma metastasis
Proteomic findings of human colorectal carcinoma SW480 and SW620 cells with differential metastatic potential provide the basis for searching potential markers for HCC prognosis and give some clues to elucidate the mechanism of CRC metastasis. To further explore the clinical significance of the proteomic finding, HSP27 was screened from comparative proteomic analysis of SW480 and SW620 cells and was found to negatively correlate with CRC metastasis in more progressively high metastatic CRC cells using Western blot, immunocytochemistry and RT-PCR analysis. Differential expression and location of HSP27 in CRC cells were further validated by immunocytochemistry technology. 68 specimens of CRC tissues were examinated by immunohistochemistry staining. HSP27 expression was not related to several clinicopathologic factor, including age, sex, histologic grade, lymphytic metastasis and clinical stage (χ~2 test, p＞0.05), but a statistically significant difference in HSP27 overexpression was found in relation to lymphytic metastasis (Fisher's exact test, p=0.035＜0.05). Our results indicate that HSP27 overexpression relates to metastatic behavior of CRC cell and probably have suppressive role in progression and malignant transformation of CRC.
In a word, the present study made a systematic research on tumor metastasis by comparative proteome technique using SW480 and SW620 as metastatic model. 11 metastasis-associated proteins were identified and characterized, more importantly, there were no clear evidence about the association of candidates HSP27 with tumor metastasis to the best of our knowledge. Function clinical significance confirmed that HSP27 overexpression relates to metastatic behavior of CRC cell and probably have suppressive role in progression of CRC. HSP27 may be a key factor for preventing tumor metastais and a therapeutic target for the treatment of CRC patients with metastasis.
肿瘤转移是一个多步骤、多基因参与的复杂过程，肿瘤转移的全过程受以下几个因素的调控：1、在转移启动初期E-cadherin、Ig超家族、选择素、整合素等参与肿瘤细胞间的脱落以及肿瘤细胞与胞外基质的粘附；2、肿瘤细胞或间质细胞分泌的蛋白水解酶及其抑制剂，在降解胞外基质和血管的基底膜过程中发挥重要作用；3、在运动因子、生长因子、归巢因子等的作用下，肿瘤细胞迁移到转移靶器官；4、血管增生因子VEGF、bFGF、IL-8、PDGF等促进新生血管的形成，伴随着肿瘤细胞的增生，形成新的转移灶，完成转移过程。鉴于目前对肿瘤转移机制的研究往往针对单个或少数几个分子以及关注基因水平的改变，缺乏系统性和综合性的分析，因此在所有这些调控因素中，究竟哪些分子在肿瘤转移中发挥关键作用，仍有待阐明。近年来开展的蛋白质组学技术为揭示肿瘤转移机制带来了新的希望。
本研究以人大肠癌高、低转移细胞株为研究对象，采用比较蛋白质组技术鉴定了11个与大肠癌转移相关的候选蛋白，并探讨了HSP27在大肠癌转移中的作用及其功能，具体研究结果如下：
1、人大肠癌细胞系蛋白质双向凝胶电泳技术的建立及其优化
优化人结肠癌Lovo细胞系的蛋白质样品制备方法，建立人结肠癌Lovo细胞系的双向凝胶电泳(2-DE)图谱，为识别鉴定结肠癌发生发展相关蛋白质奠定基础。分别用磷酸盐缓冲液和等渗蔗糖溶液冲洗细胞，胰酶消化或直接刮刀法收集细胞后提取总蛋白质，利用固相pH梯度双向凝胶电泳技术进行分离，凝胶经银染显色后，用PDQuest软件分析2-DE图谱。结果以等渗蔗糖缓冲液冲洗细胞并直接刮刀法所提取Lovo细胞总蛋白质进行双向电泳，可获得分辨率高、重复性好的人结肠癌Lovo细胞系2-DE图谱。等渗蔗糖溶液有效的降低了盐离子对等电聚焦的影响，聚焦效果优于磷酸盐缓冲溶液处理组；皿上直接刮刀收集的方法避免引入胰酶及其对细胞的破坏，保证了细胞蛋白质的完整性和代表性。我们实验表明以等渗蔗糖溶液冲洗细胞结合皿上直接裂解是一种较好的细胞蛋白质双向电泳样品制备方法，在此基础上我们初步建立了分辨率较高且重复性好的人结肠癌Lovo细胞系蛋白质双向凝胶电泳图谱。
2、人大肠癌细胞株SW480和SW620的蛋白质组双向凝胶电泳分析
为了更好的理解大肠癌转移机理和寻找大肠癌预后的标志分子，我们运用双向凝胶电泳技术对人大肠癌高、低转移细胞株SW480和SW620细胞进行了差异表达双向凝胶电泳分析，数字化图像分析发现有72个蛋白质斑点在两组凝胶中有显著差异表达，在SW620细胞中表达量升高的蛋白质斑点有30个，在SW480细胞中表达量升高的蛋白质斑点有42个；定性差异表达分析(表达量相差10倍以上)发现22个蛋白质斑点仅在SW620中出现，而有27个蛋白质斑点只在SW480中检测到，结合软件分析和手工筛选，选取15个点清晰且表达水平改变明显的蛋白质点作为质谱鉴定的靶点。
3、双向凝胶电泳差异表达蛋白质的质谱鉴定和验证
以大肠癌高、低转移细胞株为研究对象，采用2-DE分离技术结合MALDI-TOF质谱鉴定了11个与大肠癌转移相关的蛋白，其中SW620细胞株表达上调的蛋白质有磷酸甘油酸变位酶1，磷脂酰乙醇胺结合蛋白和高迁移率族蛋白B-1，而热休克蛋白27，膜联蛋白Ⅰ，甲硫腺苷磷酸化酶，切丝蛋白1和表皮型脂肪酸结合蛋白在SW620中表达下调。大多数差异蛋白功能涉及肿瘤细胞生长、运动、粘附、凋亡等过程，研究结果为阐明大肠癌转移机制及寻找预测大肠癌转移的潜在标志物提供了理论依据。
为保证蛋白质组技术鉴定结果的可靠性，在蛋白和mRNA水平对候选蛋白进行了进一步验证。采用western blot和免疫细胞化学技术在蛋白质水平验证结果表明HSP27和COF1在低转移的SW480中高表达，且COF1仅在SW480中检测到，蛋白质水平的验证结果与比较蛋白质组的分析结果完全一致。此外，采用半定量RT-PCR技术对编码候选蛋白的mRNA水平检测结果表明：编码HMGB1和PBP的mRNA在高转移的SW620中高表达，而编码HSP27、ANXⅠ、COF1和E-FABP的mRNA在SW480中高表达，所验证的编码候选蛋白的mRNA水平验证结果与候选蛋白的蛋白质组分析结果一致，说明候选蛋白在大肠癌细胞株中的蛋白水平变化主要是由于基因的转录水平高低不同所致，也肯定了候选蛋白在高、低转移株间的差异表达。结果表明通过蛋白质组技术筛选到的转移相关蛋白是可靠的。
4、热休克蛋白27与大肠癌转移的相关性分析
为更好的理解大肠癌细胞的转移机理和寻找可能的肿瘤预后标记分子标志物，我们用蛋白质组学方法对两种不同转移潜能的SW620和SW480细胞株的差异表达蛋白进行了研究，发现热休克蛋白27在高转移潜能大肠癌细胞中的低表达。免疫细胞化学进一步验证和分析了该蛋白在SW620和SW480两种细胞中的差异表达和定位。68例临床大肠癌组织标本的免疫组织化学研究发现热休克蛋白27的表达率与年龄、性别、组织学分级、淋巴结转移和临床分期无相关性(x~2 test，p＞0.05)，但其过表达和大肠癌转移显著负相关(Fisher's exact test，p=0.035＜0.05)。结果表明，大肠癌细胞热休克蛋白27的过表达和肿瘤细胞的转移行为相关，可能在阻止其转移中发挥重要的作用。
综上所述，本研究应用比较蛋白质组技术对人大肠癌SW620和SW480两种不同转移潜能细胞株进行了对比研究，获得了11个差异表达蛋白质并对HSP27进行了初步验证。分析结果证实大肠癌细胞热休克蛋白27的过表达和肿瘤细胞的转移行为相关，在阻止大肠癌转移中发挥重要的作用，可能是阻止大肠癌转移的关键分子之一，研究结果将为抗大肠癌转移研究和药物的筛选提供有用的研究信息和药物分子靶标。
Widespread metastasis is a common phenomenon and the major lethal cause of cancer. So far, however, very little is known about the mechanisms underlying metastasis. Once the key factors in tumor metastasis were screened and identified, the drug targets and diagnosis markers could be obtained accordingly.Metastasis is a complex multistep malignant process. It has been widely reported that many molecules were involved in the complex process of tumor invasion and metastasis: 1) E-cadherin, immunoglobin superfamily, selectin and integrin have been suggested to take part in the detachment of tumor cells from the primary site and interaction of tumor cells with the surrounding extracellular matrix; 2) Matrix-degrading enzymes and their inhibitors secreted by tumor cells or mesenchymal cells have been indicated to be involved in degradation of extracellular matrix and vascular basement; 3) Some growth factors and movememt factors have been implicated to play role in migration of tumor cells into secondary sites; 4) Angiogenetic factors, such as VEGF, bFGF, EL-8 and PDGF, have been demonstrated to be important in neoangiogenesis and distant metastasis. At present, most of the research associated with metastasis so far has been focused on the genetic changes of related molecules or single or few proteins without systematic study. But little is known about the key factors to trigger tumorigenic cells to initiate further invasion and metastasis facing with so many regulators up to now.
On the basis of these considerations, proteomic strategy, combined with two-dimensional electrophoresis (2-DE) separation and mass spectrometry (MS) identification with advantage of high resolution, high reproducibility was used to separate and identify differentially expressed proteins between highly and lowly metastatic subpopulations. In this study we hoped to find out a series of protein cluster deeply involved in metastasis process and addressed the question whether there were new proteins to be associated with metastasis, moreover, to clarify the metastasis-associated function mediated by new candidate proteins. In the present study, 11 metastasis-associated proteins were seperated and identified by comparative proteome technique using established metastatic model, more importantly, the metastasis-associated clinical characterization mediated by HSP27 was further characterized.
1. Establishment and optimization of two-dimensional polyacrylamide gel electrophoresis for the proteome analysis of the human colorectal cancer cell line
To optimize the sample preparation methods for the proteome analysis of human colon adenocarcinoma cell line Lovo, to establish a high resolution and reproducible two-dimensional polyacrylamide gel electrophoresis (2DE) image, and to provide a base for identifying disease-associated proteins. For cells cultured on solid substrates, the cell layer should be washed with phosphate-buffered saline or an isotonic sucrose solution respectively. The cells can then be harvested by treating with typsin, or scraping after having been lysed directly. Immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis was used to separate the total proteins of the samples. After silver staining, PDQuest image analysis software was applied to analyze 2DE images. Finally, the total proteins of ceils, washed with isotonic sucrose solution and scraped after having been lysed directly, were used to perform 2DE. 2DE patterns with high resolution and reproducibility of human colon adenocarcinoma cell line Lovo were obtained. The isotonic sucrose solution is a particularly efficient way of minimizing salts that can interfere with the IEF dimension. The method of scraping after having been lysed directly avoids the use of proteolytic enzymes that can destroy the cells, and it makes the total proteins complete and representative. Our findings indicated that washing with isotonic sucrose solution combinated with scraping after having been lysed directly is a proper method for protein sample preparation of human colon adenocarcinoma cell line Lovo. The high-quality separation of 2-DE gel was obtained. The well-resolved, reproducible 2-DE profiles of human colon adenocarcinoma cell line Lovo have been primarily established.
2. Comparative proteomic analysis between SW620 and SW480 cell lines with different metastasis potential
To better understand the mechanism underlying the CRC metastasis and to search potential markers for CRC prognosis, differential proteome analysis on two CRC cell strains with high and low metastatic potentials, SW620 and SW480, was conducted using various proteomics approaches. By two-dimensional gel electrophoresis (2-DE), image analysis and mass spectrometry, the expression levels of 72 protein spots were found quantitatively changed between SW620 and SW480, among them the expression levels of 30 protein spots were increased in SW620 cells and the expression levels of 42 protein spots were decreased in SW620 cells. Qualitative analysis between SW620 and SW480 cells found that 22 protein spots were only detected in SW620 cells and 27 protein spots in SW480 cells. Combinated with software and manual screening, 15 differentially expressed protein spots between SW620 and SW480 were further identified by MALDI-TOF MS analysis.
3. Further identification and verification of candidate proteins
Two-dimensional (2-D) gel electrophoresis followed by matrix-assisted laser desorption/time of flight mass spectrometry was utilized to compare the protein expression profiles between two colorectal cancer (CRC) cell lines with high and low metastatic potentials, SW620 and SW480. 11 metastasis-associated proteins were identified successfully. Of the identified proteins, the expressions of phosphoglycerate mutase 1, phosphatidylethanolamine binding protein and high-mobility group box 1 were elevated in SW620 cells. However, heat shock protein 27, annexinⅠ, methylthioadenosine phosphorylase, cofilin-1 and epidermal fatty acid binding protein were down-regulated in SW620 cells. Most of the candidate proteins have been evidenced to be somehow associated with various aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, etc. These results provide the basis for searching for potential markers for CRC prognosis and give some clues to elucidate the mechanism of CRC metastasis.
Western blot, immunocytochemistry and semi-quantitative RT-PCR analysis were used to further verify candidate proteins in order to ensure the reliabilty of the proteome results. The western blot and immunocytochemisty results showed that HSP27 and COF1 were significantly higher in SW480, and that COF1 was only detected in lowly metastatic SW480 cells. Evidently, western blot results were consistent with proteome analysis. In addition, most of the identified candidate proteins were further confirmed by semi-quantitative RT-PCR analysis at mRNA level. Semi-quantitative RT-PCR analysis indicated that the mRNAs coding HMGB1 and PBP were up-regulated, but the mRNAs coding HSP27, ANX I, COF1 and E-FABP were down-regulated in highly metastatic SW620 subpopulation compared with lowly metastatic SW480. The verification results indicated that most of the differences of protein expression displayed by 2-DE between SW620 and SW480 was convincing.
4. Overexpression of HSP27 correlates with colorectal carcinoma metastasis
Proteomic findings of human colorectal carcinoma SW480 and SW620 cells with differential metastatic potential provide the basis for searching potential markers for HCC prognosis and give some clues to elucidate the mechanism of CRC metastasis. To further explore the clinical significance of the proteomic finding, HSP27 was screened from comparative proteomic analysis of SW480 and SW620 cells and was found to negatively correlate with CRC metastasis in more progressively high metastatic CRC cells using Western blot, immunocytochemistry and RT-PCR analysis. Differential expression and location of HSP27 in CRC cells were further validated by immunocytochemistry technology. 68 specimens of CRC tissues were examinated by immunohistochemistry staining. HSP27 expression was not related to several clinicopathologic factor, including age, sex, histologic grade, lymphytic metastasis and clinical stage (χ~2 test, p＞0.05), but a statistically significant difference in HSP27 overexpression was found in relation to lymphytic metastasis (Fisher's exact test, p=0.035＜0.05). Our results indicate that HSP27 overexpression relates to metastatic behavior of CRC cell and probably have suppressive role in progression and malignant transformation of CRC.
In a word, the present study made a systematic research on tumor metastasis by comparative proteome technique using SW480 and SW620 as metastatic model. 11 metastasis-associated proteins were identified and characterized, more importantly, there were no clear evidence about the association of candidates HSP27 with tumor metastasis to the best of our knowledge. Function clinical significance confirmed that HSP27 overexpression relates to metastatic behavior of CRC cell and probably have suppressive role in progression of CRC. HSP27 may be a key factor for preventing tumor metastais and a therapeutic target for the treatment of CRC patients with metastasis.
引文
2003, 3(10):1988-2001.
9、 Friedman D, Hill S, Keller J,et.al. Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry. Proteomics,2004,4(3):793-811.
10、 Ding S, Li Y, Shao X,et.al. Proteome analysis of hepatocellular carcinoma cell strains, MHCC97-H and MHCC97-L, with different metastasis potentials. Proteomics, 2004,4(4):982-94.
11、 Ding S, Li Y, Tan Y,et.al. From proteomic analysis to clinical significance: overexpression of cytokeratin 19 correlates with hepatocellular carcinoma metastasis.Mol Cell Proteomics, 2004, 3(1):73-81.
12、 Jiang D, Ying W, Lu Y, et.al. Identification of metastasis-associated proteins by proteomic analysis and functional exploration of interleukin-18 in metastasis. Proteomics, 2003, 3(5):724-37.
13、 Cicek M, Samant R, Kinter M, et.al. Identification of metastasis-associated proteins through protein analysis of metastatic MDA-MB-435 and metastasis-suppressed BRMS1 transfected-MDA-MB-435 cells. Clin Exp Metastasis,2004, 21(2): 149-57.
14、 Wu W, Tang X, Hu W, et.al. Identification and validation of metastasis-associated proteins in head and neck cancer cell lines by two-dimensional electrophoresis and mass spectrometry. Clin Exp Metastasis, 2002,19(4): 319-26.
15、 Hirano T, Gong Y, Yoshida K, et.al. Usefulness of TA02 (napsin A) to distinguish primary lung adenocarcinoma from metastatic lung adenocarcinoma. Lung Cancer, 2003 , 41(2): 155-62.
16、 Ahmed N, Barker G, Oliva K, et.al. Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer. Br J Cancer, 2004, 91(1):129-40.
17、 Torres-Cabala C, Panizo-Santos A, Krutzsch H, et.al. Differential expression of S100C in thyroid lesions. Int J Surg Pathol, 2004 , 12(2):107-15.
18、 Rosty C, Christa L, Kuzdzal S, et.al. Identification of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein I as a biomarker for pancreatic ductal adenocarcinoma by protein biochip technology. Cancer Res,2002, 62(6): 1868-75.
19、 Liu A, Zhang H, Sorensen C, et.al. Analysis of prostate cancer by proteomics using tissue specimens. J Urol, 2005, 173(1):73-8.
20、 Fahrig R, Heinrich J, Nickel B, et.al. Inhibition of induced chemoresistance by cotreatment with (E)-5-(2-bromovinyl)-2'-deoxyuridine (RP101). Cancer Res,2003,63(18):5745-53.
21、 Lim Y, Wong C, Ooi L, et.al. Selective tyrosine hyperphosphorylation of cytoskeletal and stress proteins in primary human breast cancers: implications for adjuvant use of kinase-inhibitory drugs. Clin Cancer Res, 2004, 10(12 Pt 1):3980-7.
22、 Harris M, Ozpolat B, Abdi F, et.al. Comparative proteomic analysis of all-trans-retinoic acid treatment reveals systematic posttranscriptional control mechanisms in acute promyelocytic leukemia. Blood, 2004, 104(5): 1314-23. Epub 2004 May 13.
23、 Seliger B, Menig M, Lichtenfels R, et.al. Identification of markers for the selection of patients undergoing renal cell carcinoma-specific immunotherapy. Proteomics, 2003, 3(6):979-90.
24、 Oh P, Li Y, Yu J, et.al. Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for tissue-specific therapy. Nature, 2004,429(6992):629-35.
9、 Friedman D, Hill S, Keller J,et.al. Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry. Proteomics,2004,4(3):793-811.
10、 Ding S, Li Y, Shao X,et.al. Proteome analysis of hepatocellular carcinoma cell strains, MHCC97-H and MHCC97-L, with different metastasis potentials. Proteomics, 2004,4(4):982-94.
11、 Ding S, Li Y, Tan Y,et.al. From proteomic analysis to clinical significance: overexpression of cytokeratin 19 correlates with hepatocellular carcinoma metastasis.Mol Cell Proteomics, 2004, 3(1):73-81.
12、 Jiang D, Ying W, Lu Y, et.al. Identification of metastasis-associated proteins by proteomic analysis and functional exploration of interleukin-18 in metastasis. Proteomics, 2003, 3(5):724-37.
13、 Cicek M, Samant R, Kinter M, et.al. Identification of metastasis-associated proteins through protein analysis of metastatic MDA-MB-435 and metastasis-suppressed BRMS1 transfected-MDA-MB-435 cells. Clin Exp Metastasis,2004, 21(2): 149-57.
14、 Wu W, Tang X, Hu W, et.al. Identification and validation of metastasis-associated proteins in head and neck cancer cell lines by two-dimensional electrophoresis and mass spectrometry. Clin Exp Metastasis, 2002,19(4): 319-26.
15、 Hirano T, Gong Y, Yoshida K, et.al. Usefulness of TA02 (napsin A) to distinguish primary lung adenocarcinoma from metastatic lung adenocarcinoma. Lung Cancer, 2003 , 41(2): 155-62.
16、 Ahmed N, Barker G, Oliva K, et.al. Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer. Br J Cancer, 2004, 91(1):129-40.
17、 Torres-Cabala C, Panizo-Santos A, Krutzsch H, et.al. Differential expression of S100C in thyroid lesions. Int J Surg Pathol, 2004 , 12(2):107-15.
18、 Rosty C, Christa L, Kuzdzal S, et.al. Identification of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein I as a biomarker for pancreatic ductal adenocarcinoma by protein biochip technology. Cancer Res,2002, 62(6): 1868-75.
19、 Liu A, Zhang H, Sorensen C, et.al. Analysis of prostate cancer by proteomics using tissue specimens. J Urol, 2005, 173(1):73-8.
20、 Fahrig R, Heinrich J, Nickel B, et.al. Inhibition of induced chemoresistance by cotreatment with (E)-5-(2-bromovinyl)-2'-deoxyuridine (RP101). Cancer Res,2003,63(18):5745-53.
21、 Lim Y, Wong C, Ooi L, et.al. Selective tyrosine hyperphosphorylation of cytoskeletal and stress proteins in primary human breast cancers: implications for adjuvant use of kinase-inhibitory drugs. Clin Cancer Res, 2004, 10(12 Pt 1):3980-7.
22、 Harris M, Ozpolat B, Abdi F, et.al. Comparative proteomic analysis of all-trans-retinoic acid treatment reveals systematic posttranscriptional control mechanisms in acute promyelocytic leukemia. Blood, 2004, 104(5): 1314-23. Epub 2004 May 13.
23、 Seliger B, Menig M, Lichtenfels R, et.al. Identification of markers for the selection of patients undergoing renal cell carcinoma-specific immunotherapy. Proteomics, 2003, 3(6):979-90.
24、 Oh P, Li Y, Yu J, et.al. Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for tissue-specific therapy. Nature, 2004,429(6992):629-35.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |