氯离子通道蛋白-1（CLIC1）在结肠癌转移中的作用及机制研究
摘要
背景及目的
结肠癌是常见的消化道恶性肿瘤，其治疗目前仍以手术为主。虽然肿瘤被完全切除,但术后仍可能发生播散性转移，术后5年生存率较低，约50%，近半数病例术后2年出现肿瘤的复发和转移。氯离子通道蛋白-1(Chloride intracellular channel1, CLIC1)是新近发现的一种离子通道蛋白，它属于氯通道蛋白家族中的一种，最近研究表明CLIC1在肝癌、胆囊癌和胃癌等肿瘤组织中高表达，并与胆囊癌及胃癌的转移相关，在结直肠癌中也检测到CLIC1的高表达，但是CLIC1是否与结直肠癌的转移有关未作相关研究。因此本课题从以下三个方面对CLIC1与结肠癌的转移作一初步研究：(1)CLIC1在结肠癌组织及癌旁组织中的表达有无差异，CLIC1的表达结肠癌患者与患者的性别、年龄、肿瘤大小、有无淋巴结转移及TNM分期等是否有关；(2)CLIC1能否通过调控性容积下降（RVD）介导结肠癌的转移；(3)CLIC1在缺氧-再加氧条件下与结肠癌转移及其机制，从而为结肠癌的防治提供新的思路和方法。
研究方法与结果
第一部分CLIC1mRNA及蛋白在结肠癌表达及临床意义
方法：收集2011年1-6月本院手术切除的54例结肠癌和相应癌旁组织(距肿瘤边缘＞5cm)标本。采用逆转录RT-PCR检测CLIC1mRNA在结肠癌组织及相应癌旁组织中的表达情况，免疫组化检测CLIC1蛋白在结肠癌组织的表达。比较CLIC1mRNA在结肠癌及癌旁组织的表达差异，分析CLIC1蛋白的表达与患者年龄、性别、肿瘤的大小、部位、分化程度、淋巴结转移及TNM分期等方面的关系。结果：CLIC1mRNA在54例患者癌组织中表达的阳性率为85.18%(46/54)；在相应癌旁组织中表达的阳性率为90.74%(49/54)，两者CLIC1mRNA的阳性表达率之间比较无明显差异(P>0.05)。CLIC1mRNA在结肠癌及癌旁组织中的扩增水平别为0.6576±0.043和0.2808±0.027，两者比较P<0.01。结肠癌组织中CLIC1蛋白的表达水平高于癌旁组织，CLIC1蛋白表达在患者肿瘤的浸润程度(P<0.05)和有无淋巴转移(P<0.05)存在差异；在TNM分期(P<0.01)差异有显著性；而CLIC1蛋白表达在患者的年龄、性别、肿瘤的大小、部位、分化程度方面无明显差异(P>0.05)。
第二部分CLIC1介导RVD促进结肠癌迁移及侵袭
方法：我们用人结肠癌细胞株LOVO和HT29作为细胞模型来研究氯离子通道蛋白-1(CLIC1)在结肠癌转移中的作用。用低渗液分别刺激诱导LOVO和HT29细胞的调控性细胞容积下降(RVD)过程，比较两者RVD的差异；然后CLIC1特异性抑制剂IAA94观察对LOVO和HT29细胞RVD的影响。分别采用CLIC1特异性抑制剂IAA94和CLIC1SiRNA转染，研究它们对结肠癌细胞迁移及侵袭的影响。通过RT-PCR、Western Blot及细胞免疫荧光检测CLIC1mRNA及其蛋白的表达。结果：当两种细胞暴露于低渗溶液时，高转移潜能的LOVO和低转移潜能的HT29细胞细胞，与在等渗溶液中比较细胞容积分别增加45.6±1.8％(P <0.01)和46.8±2.3％(P <0.01)。在肿胀高峰后，细胞容积逐渐下降。LOVO细胞标准化细胞容积从峰值145.6±1.8％(低渗5分钟)，下降至最小值109.8±3.0％(低渗17分钟)(P <0.01)；HT29细胞标准化细胞容积，从峰值146.8±2.3％(低渗5分钟)，在低渗16分钟下降至最小值120.5±2.1％(低渗16分钟)(P <0.01)。HT29和LOVO细胞的RVD值分别为61.4±2.2％和81.09±1.9％，两者比较差异有显著性(P <0.01)。特异的氯离子通道蛋白-1阻滞剂IAA94(20或40μmol/L)可抑制结肠癌细胞的RVD、迁移和侵袭能力。这些效应呈剂量依赖性。小RNA(SiRNA)干扰下调CLIC1的表达亦可抑制LOVO和HT29细胞的迁移和侵袭能力。CLIC1蛋白均主要表达在两种类型细胞的细胞膜，表明可发挥氯离子通道功能；与HT29细胞相比，CLIC1mRNA和蛋白的表达在LOVO细胞均高表达(P <0.01)。氯离子通道蛋白-1特异阻滞剂IAA94(20或40μmol/L)对两种细胞的CLIC1mRNA和蛋白表达水平均无明显的影响。
第三部分CLIC1通过ROS/ERK信号通路介导结肠癌LOVO细胞的迁移及侵袭
方法：缺氧-再加氧(H-R)培养处理人结肠癌LOVO细胞，荧光探针DCF-DA检测检测细胞内活性氧(ROS)水平，伤口愈合及Transwell细胞侵袭试验分别检测对LOVO细胞迁移及侵袭的影响。NADPH氧化酶抑制剂(DPI)、抗氧化剂(NAC)、ERK抑制剂(PD98059)及氯离子通道蛋白-1抑制剂(IAA94)分别处理H-R条件下LOVO细胞，检测对细胞ROS产生、迁移及侵袭的影响，Western blot检对ERK、p-ERK、MMP-2及MMP-9蛋白表达的影响。结果：与常氧培养组(N)比较，H-R处理后LOVO细胞内的ROS明显提高(P <0.01)，细胞的迁移及侵袭能力明显增强(分别P <0.05，P <0.01)， p-ERK、MMP-2和MMP-9蛋白表达水平明显上调。与H-R组比较，DPI(15μmol/L)组; NAC(30mmol/L)组; PD98059(50μmol/L)组或IAA94(20和40μmol/L)组细胞ROS产生、迁移及侵袭能力明显减弱(P <0.01)， p-ERK、MMP-2和MMP-9蛋白表达水平亦明显减少。
全文结论
(1)CLIC1mRNA及其蛋白在结肠癌组织中高表达，CLIC1蛋白表达在肿瘤的浸润程度、有无淋巴转移和TNM分期方面存在显著差异；而在患者的年龄、性别、肿瘤的大小、部位、分化程度方面差异无显著性。(2)RVD与结肠癌细胞的转移潜能有关；CLIC1特异性抑制剂IAA94抑制结肠癌LOVO及HT29细胞的RVD、迁移及侵袭；CLIC1SiRNA转染两种细胞亦可抑制结肠癌细胞的迁移及侵袭能力；CLIC1可通过调控结肠癌细胞的RVD介导结肠癌的转移。(3)H-R可通过提高细胞内的ROS产生促进结肠癌的迁移及侵袭能力；CLIC1可通过ROS/ERK信号途径调控结肠癌LOVO细胞的迁移及侵袭。
Background Colon cancer is a common gastrointestinal cancer and surgery is still themain treatment for colon cancer. Although the tumor was completely resected, however,disseminated metastases may still emerge after the operation. The5-year-survival rate forthe cases with colon cancer is low, about50%, and about half of the cases have therecurrence and metastasis of tumor after2years of the postoperation. CLIC1(Chlorideintracellular channel1, CLIC1), one of the chloride channel protein family, is a recentlydiscovered ion channel protein. Recent studies have shown that CLIC1is highly expressedin hepatoma, gallbladder carcinoma and gastric cancer, and is related to the metastasis ofthe gallbladder carcinoma and gastric cancer. CLIC1also is highly expressed in colorectalcancer, but it is unkown whether CLIC1participate in the metastasis of colorectal cancer.This subjects of this study are the the following three aspects for the relationship betweenCLIC1and metastasis of colon cancer:(1) whether CLIC1expression differs in coloncancer tissue and adjacent tissues; whether CLIC1expression is related to the gender, age,tumor size, lymph node metastasis and TNM stage of patients with colon cancer.(2)whether CLIC1mediates the metastasis of colon cancer via regulatory cell volumedecrease(RVD).(3) involvements of CLIC1in the metastasis of colon cancer under theHypoxia-reoxygenation conditions of colon cancer metastasis and its possible mechanisms;our findings may provide new ideas and methods for the prevention and treatment of coloncancer.
Methods and Results:
Part1: CLIC1mRNA and protein expression in Colon Cancer and its ClinicalSignificance
Methods The samples of colon cancer and corresponding para-carcinoma tissues(fromthe tumor margin>5cm) of54cases by surgical resection in our hospital from January toJune2011were collected. Reverse Transcription PCR (RT-PCR) method was used to detectthe expression of the CLIC1mRNA in colon cancer tissues and corresponding adjacent-carcinoma tissues, and immunohistochemical staining was used to examine theCLIC1protein expression in colon carcinoma tissues, respectively. The comparisonbetween the CLIC1mRNA in colon cancer and adjacent-carcinoma tissues was performed,and the relationship between the CLIC1protein expression and the age, gender, tumor size,tumor location, degree of tumor differentiation, lymph node metastasis and TNM stage ofthe cases was analyzed. Results The positive rate of CLIC1mRNA expression in carcinomatissues of54cases was85.18%(46/54); the positive rate expression in the correspondingadjacent-carcinoma tissues of54cases was90.74%(49/54). There was no significantdifference between the two positive expression rates of CLIC1mRNA (P>0.05). CLIC1mRNA in colon cancer tissue was higher than that in corresponding para-carcinoma tissues(P<0.05). CLIC1mRNA level of amplification in colon cancer and adjacent-carcinomatissues is0.6576±0.043and0.2808±0.027, respectively (P <0.01).The level of CLIC1protein in tumor tissues is higer than that in adjacent-tumor tissues(P<0.01).There wasdifference among invasion degree(P <0.05), lymph node metastasis(P <0.05) and TNMstage(P <0.01) in CLIC1protein expression of tumor in patients. However, there was nosignificant difference among age, sex and the size, position and differentiation in CLIC1protein expression of tumor in patients (P>0.05, respectively).
Part2: Regulation of colon cancer cell migration and invasion by CLIC1-mediatedRVD
Methods We used the human colon cancer cell lines LOVO and HT29as modelsystems to determine the role of the chloride intracellular channel1(CLIC1) in themetastasis of colonic cancer.Hypotonic solutions were used to stimulate and induce theprocess of Regulatory volume decrease (RVD) in LOVO and HT29cells. The difference ofRVD between the two cell lines was compared. The effect of CLIC1on the RVD process inboth LOVO and HT29cells was investigated by using CLIC1specific inhibitor IAA94.CLIC1specific inhibitor IAA94and CLIC1SiRNA transfection were used to study theireffects on colon cancer cell migration and invasion. The CLIC1mRNA and its proteinexpression in both LOVO and HT29cells were detected by Reverse TranscriptasePolymerase Chain Reaction (RT-PCR), Western blot and immunofluorescence staining.Results In highly metastatic LOVO and poorly metastatic HT29cells exposed to thehypotonic solutions, the cell volumes increased by45.6±1.8%(P <0.01) and by46.8±2.3 %(P <0.01) compared with those in isotonic solution, respectively. Following the swellingpeak; cell volume decreased gradually. In LOVO cells, the standardized volume decreasedfrom a peak of145.6±1.8%(Hypo5min) to109.8±3.0%(Hypo17min)(P <0.01). InHT29cells, the standardized volume decreased from a peak of146.8±2.3%(Hypo5min)to120.5±2.1%(Hypo16min)(P <0.01) while still exposed to the hypotonic solution.The RVD rate in HT29cells (61.4±2.2%) was lower than that in LOVO cells (81.09±1.9%)(P <0.01).Functionally suppressing CLIC1using the specific chloride intracellularchannel1blocker Indanyloxyacetic acid94(IAA94) at the concentration of20μM or40μMinhibited RVD and decreased the migration and invasion of colon cancer cells. Moreover,these effects occurred in a dose-dependent manner. The migration and invasion abilities intwo cell lines were also inhibited by the knockdown of CLIC1using small interfering RNA(siRNA) transfection. In human colon cancer cells, CLIC1is primarily located in theplasma membrane，where it function as a chloride channel. Compared to HT29cells, themRNA and protein expression of CLIC1are up-regulated in LOVO cells(P <0.01). Noeffect of IAA94(20μM or40μM)on the CLIC1mRNA and its protein expression of CLIC1in LOVO or HT29cells was observed after treatment.
Part3: CLIC1regulates migration and invasion of colon cancer LOVO cells throughROS/ERK pathway
Methods After Hypoxia-reoxygenation (H-R) treatment for human colon cancerLOVO cells, fluorescent probes DCF-DA was used to detect the intracellular R eactiveoxygen species (ROS) level of LOVO cells, and wound healing and Transwell cell invasionexperiments were performed to investigate the LOVO cells migration and and invasion.Inhibitors of NADPH oxidase (DPI); antioxidant (NAC); ERK (PD98059) and Chlorideintracellular channel1(IAA94) were used to treat LOVO cells under H-R conditions,respectively. The effects of the inhibitors on intracellular ROS production, cell migrationand invasion were determined. The effects of the inhibitors on the proteins of p-ERK,MMP-2and MMP-9also were detected by Western blot analysis. Results Compared withthe normoxia group (N), ROS production in H-R group of LOVO cells was significantlyincreased (P <0.01), and cell migration and invasion capacities were significantly enhanced(P <0.05, P <0.01, respectively). The levels of p-ERK, MMP-2and MMP-9protein weresignificantly elevated under H-R conditions. Compared with the H-R group, the DPI(15μM) group; NAC(30mM) group; PD98059(50μM) group or IAA94(20and40μm) group,cellular ROS generation, cell migration and invasion capacities were significantlydecreased (P <0.01), and the protein levels of p-ERK, MMP-2and MMP-9were alsosignificantly decreased(P <0.01).
Conclusion
(1) CLIC1mRNA and its protein are highly expressed in colon cancer tissues, andCLIC1protein expression of CLIC1protein was significantly different in the tumorinvasion degree, lymph node metastasis and TNM stage of the patients; however, there wasno significant difference among age, sex and the size, position and differentiation in CLIC1protein expression of tumors in patients.(2) RVD is related with the metastatic potential ofcolon cancer cells; the CLIC1specific inhibitor IAA94inhibits RVD, migration andinvasion of the colon LOVO and HT29cells; the CLIC1SiRNA transfection with two celltypes can also inhibit colon cancer cell migration and invasiion abilities; CLIC1-mediatedRVD is involved in metastasis of colon cancer.(3) H-R can promote the cell migration andinvasion of colon cancer by increasing the intracellular ROS production; CLIC1regulatesthe colon LOVO cell migration and invasion through ROS/ERK pathway.
结肠癌是常见的消化道恶性肿瘤，其治疗目前仍以手术为主。虽然肿瘤被完全切除,但术后仍可能发生播散性转移，术后5年生存率较低，约50%，近半数病例术后2年出现肿瘤的复发和转移。氯离子通道蛋白-1(Chloride intracellular channel1, CLIC1)是新近发现的一种离子通道蛋白，它属于氯通道蛋白家族中的一种，最近研究表明CLIC1在肝癌、胆囊癌和胃癌等肿瘤组织中高表达，并与胆囊癌及胃癌的转移相关，在结直肠癌中也检测到CLIC1的高表达，但是CLIC1是否与结直肠癌的转移有关未作相关研究。因此本课题从以下三个方面对CLIC1与结肠癌的转移作一初步研究：(1)CLIC1在结肠癌组织及癌旁组织中的表达有无差异，CLIC1的表达结肠癌患者与患者的性别、年龄、肿瘤大小、有无淋巴结转移及TNM分期等是否有关；(2)CLIC1能否通过调控性容积下降（RVD）介导结肠癌的转移；(3)CLIC1在缺氧-再加氧条件下与结肠癌转移及其机制，从而为结肠癌的防治提供新的思路和方法。
研究方法与结果
第一部分CLIC1mRNA及蛋白在结肠癌表达及临床意义
方法：收集2011年1-6月本院手术切除的54例结肠癌和相应癌旁组织(距肿瘤边缘＞5cm)标本。采用逆转录RT-PCR检测CLIC1mRNA在结肠癌组织及相应癌旁组织中的表达情况，免疫组化检测CLIC1蛋白在结肠癌组织的表达。比较CLIC1mRNA在结肠癌及癌旁组织的表达差异，分析CLIC1蛋白的表达与患者年龄、性别、肿瘤的大小、部位、分化程度、淋巴结转移及TNM分期等方面的关系。结果：CLIC1mRNA在54例患者癌组织中表达的阳性率为85.18%(46/54)；在相应癌旁组织中表达的阳性率为90.74%(49/54)，两者CLIC1mRNA的阳性表达率之间比较无明显差异(P>0.05)。CLIC1mRNA在结肠癌及癌旁组织中的扩增水平别为0.6576±0.043和0.2808±0.027，两者比较P<0.01。结肠癌组织中CLIC1蛋白的表达水平高于癌旁组织，CLIC1蛋白表达在患者肿瘤的浸润程度(P<0.05)和有无淋巴转移(P<0.05)存在差异；在TNM分期(P<0.01)差异有显著性；而CLIC1蛋白表达在患者的年龄、性别、肿瘤的大小、部位、分化程度方面无明显差异(P>0.05)。
第二部分CLIC1介导RVD促进结肠癌迁移及侵袭
方法：我们用人结肠癌细胞株LOVO和HT29作为细胞模型来研究氯离子通道蛋白-1(CLIC1)在结肠癌转移中的作用。用低渗液分别刺激诱导LOVO和HT29细胞的调控性细胞容积下降(RVD)过程，比较两者RVD的差异；然后CLIC1特异性抑制剂IAA94观察对LOVO和HT29细胞RVD的影响。分别采用CLIC1特异性抑制剂IAA94和CLIC1SiRNA转染，研究它们对结肠癌细胞迁移及侵袭的影响。通过RT-PCR、Western Blot及细胞免疫荧光检测CLIC1mRNA及其蛋白的表达。结果：当两种细胞暴露于低渗溶液时，高转移潜能的LOVO和低转移潜能的HT29细胞细胞，与在等渗溶液中比较细胞容积分别增加45.6±1.8％(P <0.01)和46.8±2.3％(P <0.01)。在肿胀高峰后，细胞容积逐渐下降。LOVO细胞标准化细胞容积从峰值145.6±1.8％(低渗5分钟)，下降至最小值109.8±3.0％(低渗17分钟)(P <0.01)；HT29细胞标准化细胞容积，从峰值146.8±2.3％(低渗5分钟)，在低渗16分钟下降至最小值120.5±2.1％(低渗16分钟)(P <0.01)。HT29和LOVO细胞的RVD值分别为61.4±2.2％和81.09±1.9％，两者比较差异有显著性(P <0.01)。特异的氯离子通道蛋白-1阻滞剂IAA94(20或40μmol/L)可抑制结肠癌细胞的RVD、迁移和侵袭能力。这些效应呈剂量依赖性。小RNA(SiRNA)干扰下调CLIC1的表达亦可抑制LOVO和HT29细胞的迁移和侵袭能力。CLIC1蛋白均主要表达在两种类型细胞的细胞膜，表明可发挥氯离子通道功能；与HT29细胞相比，CLIC1mRNA和蛋白的表达在LOVO细胞均高表达(P <0.01)。氯离子通道蛋白-1特异阻滞剂IAA94(20或40μmol/L)对两种细胞的CLIC1mRNA和蛋白表达水平均无明显的影响。
第三部分CLIC1通过ROS/ERK信号通路介导结肠癌LOVO细胞的迁移及侵袭
方法：缺氧-再加氧(H-R)培养处理人结肠癌LOVO细胞，荧光探针DCF-DA检测检测细胞内活性氧(ROS)水平，伤口愈合及Transwell细胞侵袭试验分别检测对LOVO细胞迁移及侵袭的影响。NADPH氧化酶抑制剂(DPI)、抗氧化剂(NAC)、ERK抑制剂(PD98059)及氯离子通道蛋白-1抑制剂(IAA94)分别处理H-R条件下LOVO细胞，检测对细胞ROS产生、迁移及侵袭的影响，Western blot检对ERK、p-ERK、MMP-2及MMP-9蛋白表达的影响。结果：与常氧培养组(N)比较，H-R处理后LOVO细胞内的ROS明显提高(P <0.01)，细胞的迁移及侵袭能力明显增强(分别P <0.05，P <0.01)， p-ERK、MMP-2和MMP-9蛋白表达水平明显上调。与H-R组比较，DPI(15μmol/L)组; NAC(30mmol/L)组; PD98059(50μmol/L)组或IAA94(20和40μmol/L)组细胞ROS产生、迁移及侵袭能力明显减弱(P <0.01)， p-ERK、MMP-2和MMP-9蛋白表达水平亦明显减少。
全文结论
(1)CLIC1mRNA及其蛋白在结肠癌组织中高表达，CLIC1蛋白表达在肿瘤的浸润程度、有无淋巴转移和TNM分期方面存在显著差异；而在患者的年龄、性别、肿瘤的大小、部位、分化程度方面差异无显著性。(2)RVD与结肠癌细胞的转移潜能有关；CLIC1特异性抑制剂IAA94抑制结肠癌LOVO及HT29细胞的RVD、迁移及侵袭；CLIC1SiRNA转染两种细胞亦可抑制结肠癌细胞的迁移及侵袭能力；CLIC1可通过调控结肠癌细胞的RVD介导结肠癌的转移。(3)H-R可通过提高细胞内的ROS产生促进结肠癌的迁移及侵袭能力；CLIC1可通过ROS/ERK信号途径调控结肠癌LOVO细胞的迁移及侵袭。
Background Colon cancer is a common gastrointestinal cancer and surgery is still themain treatment for colon cancer. Although the tumor was completely resected, however,disseminated metastases may still emerge after the operation. The5-year-survival rate forthe cases with colon cancer is low, about50%, and about half of the cases have therecurrence and metastasis of tumor after2years of the postoperation. CLIC1(Chlorideintracellular channel1, CLIC1), one of the chloride channel protein family, is a recentlydiscovered ion channel protein. Recent studies have shown that CLIC1is highly expressedin hepatoma, gallbladder carcinoma and gastric cancer, and is related to the metastasis ofthe gallbladder carcinoma and gastric cancer. CLIC1also is highly expressed in colorectalcancer, but it is unkown whether CLIC1participate in the metastasis of colorectal cancer.This subjects of this study are the the following three aspects for the relationship betweenCLIC1and metastasis of colon cancer:(1) whether CLIC1expression differs in coloncancer tissue and adjacent tissues; whether CLIC1expression is related to the gender, age,tumor size, lymph node metastasis and TNM stage of patients with colon cancer.(2)whether CLIC1mediates the metastasis of colon cancer via regulatory cell volumedecrease(RVD).(3) involvements of CLIC1in the metastasis of colon cancer under theHypoxia-reoxygenation conditions of colon cancer metastasis and its possible mechanisms;our findings may provide new ideas and methods for the prevention and treatment of coloncancer.
Methods and Results:
Part1: CLIC1mRNA and protein expression in Colon Cancer and its ClinicalSignificance
Methods The samples of colon cancer and corresponding para-carcinoma tissues(fromthe tumor margin>5cm) of54cases by surgical resection in our hospital from January toJune2011were collected. Reverse Transcription PCR (RT-PCR) method was used to detectthe expression of the CLIC1mRNA in colon cancer tissues and corresponding adjacent-carcinoma tissues, and immunohistochemical staining was used to examine theCLIC1protein expression in colon carcinoma tissues, respectively. The comparisonbetween the CLIC1mRNA in colon cancer and adjacent-carcinoma tissues was performed,and the relationship between the CLIC1protein expression and the age, gender, tumor size,tumor location, degree of tumor differentiation, lymph node metastasis and TNM stage ofthe cases was analyzed. Results The positive rate of CLIC1mRNA expression in carcinomatissues of54cases was85.18%(46/54); the positive rate expression in the correspondingadjacent-carcinoma tissues of54cases was90.74%(49/54). There was no significantdifference between the two positive expression rates of CLIC1mRNA (P>0.05). CLIC1mRNA in colon cancer tissue was higher than that in corresponding para-carcinoma tissues(P<0.05). CLIC1mRNA level of amplification in colon cancer and adjacent-carcinomatissues is0.6576±0.043and0.2808±0.027, respectively (P <0.01).The level of CLIC1protein in tumor tissues is higer than that in adjacent-tumor tissues(P<0.01).There wasdifference among invasion degree(P <0.05), lymph node metastasis(P <0.05) and TNMstage(P <0.01) in CLIC1protein expression of tumor in patients. However, there was nosignificant difference among age, sex and the size, position and differentiation in CLIC1protein expression of tumor in patients (P>0.05, respectively).
Part2: Regulation of colon cancer cell migration and invasion by CLIC1-mediatedRVD
Methods We used the human colon cancer cell lines LOVO and HT29as modelsystems to determine the role of the chloride intracellular channel1(CLIC1) in themetastasis of colonic cancer.Hypotonic solutions were used to stimulate and induce theprocess of Regulatory volume decrease (RVD) in LOVO and HT29cells. The difference ofRVD between the two cell lines was compared. The effect of CLIC1on the RVD process inboth LOVO and HT29cells was investigated by using CLIC1specific inhibitor IAA94.CLIC1specific inhibitor IAA94and CLIC1SiRNA transfection were used to study theireffects on colon cancer cell migration and invasion. The CLIC1mRNA and its proteinexpression in both LOVO and HT29cells were detected by Reverse TranscriptasePolymerase Chain Reaction (RT-PCR), Western blot and immunofluorescence staining.Results In highly metastatic LOVO and poorly metastatic HT29cells exposed to thehypotonic solutions, the cell volumes increased by45.6±1.8%(P <0.01) and by46.8±2.3 %(P <0.01) compared with those in isotonic solution, respectively. Following the swellingpeak; cell volume decreased gradually. In LOVO cells, the standardized volume decreasedfrom a peak of145.6±1.8%(Hypo5min) to109.8±3.0%(Hypo17min)(P <0.01). InHT29cells, the standardized volume decreased from a peak of146.8±2.3%(Hypo5min)to120.5±2.1%(Hypo16min)(P <0.01) while still exposed to the hypotonic solution.The RVD rate in HT29cells (61.4±2.2%) was lower than that in LOVO cells (81.09±1.9%)(P <0.01).Functionally suppressing CLIC1using the specific chloride intracellularchannel1blocker Indanyloxyacetic acid94(IAA94) at the concentration of20μM or40μMinhibited RVD and decreased the migration and invasion of colon cancer cells. Moreover,these effects occurred in a dose-dependent manner. The migration and invasion abilities intwo cell lines were also inhibited by the knockdown of CLIC1using small interfering RNA(siRNA) transfection. In human colon cancer cells, CLIC1is primarily located in theplasma membrane，where it function as a chloride channel. Compared to HT29cells, themRNA and protein expression of CLIC1are up-regulated in LOVO cells(P <0.01). Noeffect of IAA94(20μM or40μM)on the CLIC1mRNA and its protein expression of CLIC1in LOVO or HT29cells was observed after treatment.
Part3: CLIC1regulates migration and invasion of colon cancer LOVO cells throughROS/ERK pathway
Methods After Hypoxia-reoxygenation (H-R) treatment for human colon cancerLOVO cells, fluorescent probes DCF-DA was used to detect the intracellular R eactiveoxygen species (ROS) level of LOVO cells, and wound healing and Transwell cell invasionexperiments were performed to investigate the LOVO cells migration and and invasion.Inhibitors of NADPH oxidase (DPI); antioxidant (NAC); ERK (PD98059) and Chlorideintracellular channel1(IAA94) were used to treat LOVO cells under H-R conditions,respectively. The effects of the inhibitors on intracellular ROS production, cell migrationand invasion were determined. The effects of the inhibitors on the proteins of p-ERK,MMP-2and MMP-9also were detected by Western blot analysis. Results Compared withthe normoxia group (N), ROS production in H-R group of LOVO cells was significantlyincreased (P <0.01), and cell migration and invasion capacities were significantly enhanced(P <0.05, P <0.01, respectively). The levels of p-ERK, MMP-2and MMP-9protein weresignificantly elevated under H-R conditions. Compared with the H-R group, the DPI(15μM) group; NAC(30mM) group; PD98059(50μM) group or IAA94(20and40μm) group,cellular ROS generation, cell migration and invasion capacities were significantlydecreased (P <0.01), and the protein levels of p-ERK, MMP-2and MMP-9were alsosignificantly decreased(P <0.01).
Conclusion
(1) CLIC1mRNA and its protein are highly expressed in colon cancer tissues, andCLIC1protein expression of CLIC1protein was significantly different in the tumorinvasion degree, lymph node metastasis and TNM stage of the patients; however, there wasno significant difference among age, sex and the size, position and differentiation in CLIC1protein expression of tumors in patients.(2) RVD is related with the metastatic potential ofcolon cancer cells; the CLIC1specific inhibitor IAA94inhibits RVD, migration andinvasion of the colon LOVO and HT29cells; the CLIC1SiRNA transfection with two celltypes can also inhibit colon cancer cell migration and invasiion abilities; CLIC1-mediatedRVD is involved in metastasis of colon cancer.(3) H-R can promote the cell migration andinvasion of colon cancer by increasing the intracellular ROS production; CLIC1regulatesthe colon LOVO cell migration and invasion through ROS/ERK pathway.
引文
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19. Yamaguchi H, Wyckoff J, Condeelis J. Cell migration in tumors[J]. Curr Opin Cell Biol,2005,17(5):559-564.
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