The Study Of CLIC1Involved In The Metastasis Of Colon Cancer And Its Mechanisms

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 ClinicalSignificanceMethods 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-mediatedRVDMethods 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 pathwayMethods 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.