Colorectal Carcinoma Cell Line SW480 Research Articles

Hyal1 Expression in Colorectal Carcinoma Cell Migration and Invasiveness: Significance and Mechanism.

Objective To clarify the significance of hyaluronidsase 1 (Hyal1) expression in colorectal carcinoma (CRC) and its impact on tumor cell migration and invasiveness. Methods Human CRC cell lines SW480, HCT116, and SW620 were purchased, ELISA and western blot were used to detect the expression of Hyal1 in cells, CCK-8 assay to detect cell proliferation ability, cell scratch assay to check cell migration rate, and cell invasion was detected by the transwell assay. The correlation of Hyal1 with CRC cell migration and invasiveness capacities was analyzed. Result ELISA results showed that supernatant Hyal1 level was the lowest in SW480, highest in HCT116, with the level in SW620 in between (P < 0.05). No evident difference was identified by western blot in Hyal1 protein expression among the three cells (P > 0.05). The cell scratch assay and transwell assay showed that the migration and invasion ability of HCT116 cells was higher than that of SW620 (P < 0.05). In vitro, Hyal1 had a synergistic relationship with the invasiveness and migration capacities of CRC cells (P < 0.05). Conclusion Hyal1 is elevated in CRC and is consistent with the invasiveness and metastasis abilities of CRC cells. It is hoped that this research can provide reference for future prevention and treatment of CRC.

Down-regulation of miR-29a facilitates apoptosis of colorectal carcinoma cell SW480 and suppresses its Paclitaxel resistance.

PTEN can suppress PI3K/AKT activity, and regulate cell proliferation, apoptosis, and drug resistance. The previous study showed that up-regulation of miR-29a played an essential role in the occurrence of colorectal carcinoma. This study aimed to investigate the role and related mechanism of miR-29a in mediating Paclitaxel sensitivity of colorectal carcinoma cells. Bioinformatics analysis was performed to study the existence of binding sites between miR-29a and targeting gene mRNA. Dual luciferase reporter assay was conducted to validate the targeted regulation. The expressions were compared between CCD841 CoN cells, colorectal carcinoma cell line SW480, and drug-resistant cell line SW480/Paclitaxel. Cell apoptosis and proliferation were measured by flow cytometry. In vitro cultured SW480/Paclitaxel cells were transfected with miR-29a or pcDNA3.1-PTEN. MiR-29a and PTEN expressions were measured by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and Western blot, followed by flow cytometry on the detection of cell apoptosis as well as proliferation assay. A targeted regulatory relationship existed between miR-29a and PTEN. Comparing to CCD841 CoN cells, high level of miR-29a and decreasing expression of PTEN were found in SW480 cells. Moreover, further higher miR-29a and lower PTEN expressions were observed in SW480/Paclitaxel cells. Paclitaxel remarkably inhibited proliferation and facilitated apoptosis of SW480 cells but not SW480/Paclitaxel cells. Transfection of miR-29a inhibitor or pcDNA3.1-PTEN remarkably elevated PTEN expression, suppressed p-AKT expression, weakened proliferation, and enhanced apoptosis of SW480/Paclitaxel cells. Our data demonstrated that suppression of miR-29a enhanced PTEN expression, inhibited cancer cell proliferation, facilitated apoptosis, and weakened drug resistance, which provides academic basis for the treatment of colorectal cancer.

Gene editing of the extra domain A positive fibronectin in various tumors, amplified the effects of CRISPR/Cas system on the inhibition of tumor progression.

BackgroundThe low efficiency of clustered, regularly interspaced, palindromic repeats-associated Cas (CRISPR/Cas) system editing genes in vivo limits the application. A components of the extracellular matrix (ECM), the extra domain A positive fibronectin (EDA+FN), may be a target for CRISPR/Cas system for the pro-oncogenic effects. The exclusion of EDA exon would alter the microenvironment and inhibit tumor progression, even the frequency of gene editing is still limited.ResultsThe pro-oncogenic effects were confirmed by the exclusion of EDA exon from the fibronectin gene, as illustrated by the down-regulated proliferation, migration and invasion of CNE-2Z or SW480 cells (P<0.05). Furthermore, although the efficacy of EDA exon knockout through CRISPR/Cas system was shown to be low in vivo, the EDA+FN protein levels decrease obviously, inhibiting the tumor growth rate significantly (P<0.05), which was accompanied by a decrease in Ki-67 expression and microvessel numbers, and increased E-cadherin or decreased Vimentin expression (P<0.05).Methods and materialsHuman nasopharyngeal carcinoma cell line CNE-2Z, and the colorectal carcinoma cell line SW480 were transfected with CRISPR/Cas9 plasmids targeting EDA exon. The effects of the exclusion of EDA on the cell proliferation, motility and epithelial-mesenchymal transition (EMT) were investigated, and the western blot and real-time PCR were performed to analyze the underlying mechanisms. Furthermore, CRISPR/Cas9 plasmids were injected into xenograft tumors to knockout EDA exon in vivo, and tumor growth, cell proliferation, EMT rate, or vascularization were investigated using western blot, PCR and immunohistochemistry.ConclusionCRISPR/Cas system targeting ECM components was shown to be an effective method for the inhibition of tumor progression, as these paracrine or autocrine molecules are necessary for various tumor cells. This may represent a novel strategy for overcoming the drug evasion or resistance, in addition, circumventing the low efficiency of CRISPR/Cas system in vivo.

Circadian gene hClock enhances proliferation and inhibits apoptosis of human colorectal carcinoma cells invitro and invivo.

Colorectal carcinoma (CRC) is one of the most prevalent types of malignancy-associated mortality worldwide. Previous studies have demonstrated that amplification and overexpression of the human circadian locomotor output cycles kaput gene (hClock) was closely associated with a high risk for CRC as well as poor prognosis in CRC patients. However, the underlying molecular mechanisms of CRC remain to be fully elucidated. In the present study, hClock was exogenously overexpressed in the CRC cell line SW480 via infection of a lentivirus vector expressing hClock; in addition, a lentivirus vector-based RNA interference approach, using short hairpin RNA, was performed in order to knockdown hClock in SW620 cells. The results showed that upregulation of hClock promoted proliferation and inhibited apoptosis in SW480 cells in vitro and in vivo, while downregulation of hClock inhibited SW620 cell proliferation and accelerated apoptosis in vitro. Upregulation of hClock enhanced the activity of the anti-apoptotic gene phosphorpylated (p-) AKT and inhibited the expression of the pro-apoptotic gene B cell lymphoma-2 (Bcl-2)-associated X protein and Bcl-2 homology 3 interacting domain death agonist. Furthermore, targeted inhibition of hClock activity reduced p-AKT expression. In conclusion, the results of the present study suggested that the circadian gene hClock promoted CRC progression and inhibit tumor cell apoptosis in vitro and in vivo, while silencing hClock was able to reverse this effect.

SERPINA3K induces apoptosis in human colorectal cancer cells via activating the Fas/FasL/caspase‐8 signaling pathway

SERPINA3K, also known as kallikrein-binding protein (KBP), is a serine proteinase inhibitor with anti-inflammatory and anti-angiogenic activities. Our previous studies showed that SERPINA3K inhibited proliferation in a dose-dependent manner and induced apoptosis of endothelial cells but had no influence on SGC-7901 gastric carcinoma cells or HepG2 hepatocarcinoma cells. However, it is unknown whether SERPINA3K has a direct impact on other carcinoma cells and which mechanisms are involved. In this study, we report for the first time that SERPINA3K not only decreased cell viability but also induced apoptosis in the colorectal carcinoma cell lines SW480 and HT-29. SERPINA3K-induced apoptosis of SW480 and HT-29 was rescued by interference with Fas ligand (FasL) small hairpin RNA. Moreover, SERPINA3K increased the expression of FasL and activated caspase-8. Peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor of FasL, was also upregulated by SERPINA3K in a dose-dependent manner. The upregulation effect of FasL induced by SERPINA3K was reversed after interference with PPARγ small interfering RNA. These results demonstrated that SERPINA3K-induced SW480 and HT-29 cell apoptosis was mediated by the PPARγ/Fas/FasL signaling pathway. Therefore, our study provides additional insight into the direct anti-tumor function by inducing tumor cell apoptosis of SERPINA3K in colorectal tumors.

SiRNA-mediated silencing of paxillin down-regulates ERK1/2 signaling and alters cell ultrastructure in colorectal carcinoma cell line SW480

SiRNA-mediated silencing of paxillin down-regulates ERK1/2 signaling and alters cell ultrastructure in colorectal carcinoma cell line SW480

IL-23/IL-23R: potential mediator of intestinal tumor progression from adenomatous polyps to colorectal carcinoma

Interleukin 23 (IL-23) affects tumor growth by regulating Th cells and plays a vital role in immunosuppression in tumor tissues. However, whether tumor cells are IL-23R positive or whether IL-23 has the potential to influence the growth of cancer cells directly remains unclear. The aim of this study was to clarify the molecular expression patterns of IL-23, IL-23R, and Forkhead box P3 (FOXP3) in normal tissues adjacent to cancer, in intestinal polyps (IP), and in colorectal carcinoma (CRC), and to infer the relationship between the expression patterns of these three molecules and the progress of intestinal tumors from adenomatous polyps to colorectal cancer. The levels of IL-23A, IL-23R, and FOXP3 were evaluated in normal tissues adjacent to cancer (NT, n = 13), IP (n = 26), and CRC (n = 13) using real-time PCR, ELISA, western blotting, immunohistochemistry, and immunocytochemistry. The expression of IL-23 and FOXP3 increased progressively from NT through to CRC. Immunohistochemistry staining showed that IL-23R was highly positive in carcinoma cells of the CRC group, whereas it was partially positive in cells of other groups. In addition, the human CRC cell line SW-480 exhibited weak IL-23R immunocytochemical positivity. We propose that the IL-23/IL-23R pathway is a potential route to facilitate the malignant progression of cancers. The relationship between IL-23 and FOXP3 in the microenvironment of carcinoma led us to deduce that these two molecules may interact with each other. Although the exact mechanism underlying this interaction remains a mystery, we are convinced that these two molecules are relevant in cancer progression and that IL-23 could be a potential target for cancer immunotherapy.

Endogenous Axon Guiding Chemorepulsant Semaphorin-3F Inhibits the Growth and Metastasis of Colorectal Carcinoma

To elucidate the role of Semaphorin-3F (SEMA3F), originally described as an axon guiding chemorepulsant implicated in nerve development, in the progression of colorectal carcinoma. SEMA3F and its receptor NRP2 were examined in 72 cases of human colorectal carcinoma specimens and cell lines LoVo, SW480, and SW620 with immunohistochemistry and Western blotting. SEMA3F mRNA expression in the frozen tissue specimens and cell lines was examined with quantitative reverse transcriptase-PCR. Confocal laser scanning microscopy was used for detection of cellular localization of the proteins by immunofluorescent staining. MTT assay, flow cytometry, cell adhesion and migration, and xenografts were used to evaluate biological significance of SEMA3F. SEMA3F was significantly reduced in colorectal carcinoma tissues and cell lines. Overexpression of SEMA3F resulted in reduced proliferation, adhesion to fibronectin, and migratory capability as well as reduced S-phase population and integrin αvβ3 expression of SW480 colon cancer cells. In addition, SEMA3F-overexpressing cells exhibited diminished tumorigenesis when transplanted orthotopically in nude mice and reduced liver metastases. Moreover, transfection of siRNA targeting SEMA3F in colon cancer cells increased their tumorigenicity in vivo. Endogenous SEMA3F acts as a suppressor of the growth and metastasis of human colorectal cancer cells.

Shikonin down-regulates CXCR4 expression and inhibits CXCL12-induced migratory responses in colorectal carcinoma cell line SW480

To investigate the effects of shikonin on the proliferation, expression of CXCR4 and the migratory responses to CXCL12 in colorectal carcinoma cell line SW480. The proliferation of SW480 cells was assessed by MTT assay. Cell surface expression of CXCR4 was determined by flow cytometry. The migratory ability was determined by Transwell. Shikonin inhibited the proliferation of SW480 cells in time- and concentration-dependent manner. The expression rate of CXCR4 in SW480 cells was 99.1%. After application of shikonin 0.01 micromol/L, 0.1 micromol/L and 1.0 micromol/L for 24 h, the expression rate of CXCR4 decreased to 76.0%, 59.1% and 35.5% respectively (F=1098.041, P <0.001), and the CXCL12-induced SW480 cell migratory inhibition rate was 25.2%, 38.5% and 55.7% respectively (F=48.970, P <0.001). Besides having inhibiting tumor cell proliferation effect, Shikonin may also play a role in anti-metastasis via down-regulating the expression of CXCR4 and reducing the CXCL12-induced migratory response in colorectal carcinoma cell.

Role of CCL21/CCR7 in invasion of colorectal carcinoma cell line SW480

Chemokine receptor CCR7 is up-regulated in gastrointestinal carcinomas and is significantly associated with lymphatic invasion and lymph node metastasis. This study was to investigate the role and mechanism of CCL21/CCR7 in invasion of colorectal carcinoma cell line SW480. The invasive capacity of SW480 cells was examined using Wound healing assay and Transwell assay. Expression of matrix metalloproteinase-9 (MMP-9) was measured by Western blot. SW480 cells were pre-incubated with CCL21 for 2 h before exposure to VP-16 (20 ng/mL). Cell proliferation was measured using MTT assay. Cell apoptosis was analyzed by flow cytometry and Hoechst33258 staining. Compared to the control group, more cells in the CCL21 treatment group migrated into the gap at same time points; the count of SW480 cells penetrating through the membrane after the treatment of 100ng/mL CCL21 increased significantly [(113+/-7) vs. (48+/-4)] (P<0.05); and the relative expression of MMP-9 in the CCL21 treatment group was enhanced evidently [(0.83+/-0.02) vs. (0.38+/-0.01)] (P<0.05). Although CCL21 alone did not promote proliferation of SW480 cells, pre-incubation of cells with 100ng/mL CCL21 attenuated the inhibitory effect of VP-16 on proliferation of SW480 from 68.3% to 47.4%, and reduced the apoptotic rate from (65.2+/-5.2)% to (48.7+/-3.1)%. CCL21 enhances the invasive ability of SW480 cells, induces MMP-9 expression, and promotes the survival of SW480 cells under the suboptimal circumstance in vitro.

Suppression of N-Myc Downstream-Regulated Gene 2 Is Associated with Induction of Myc in Colorectal Cancer and Correlates Closely with Differentiation

NDRG2, a new member of the N-Myc downstream-regulated gene (NDRG) family, is a focus for study at present. Up to now, its expression and function in carcinoma remain to be elucidated. In this study, using a colorectal cancer tissue array and a series of 213 colorectal cancer samples, the relationship between Ndrg2 and c-MYC expression and tumor differentiation level was investigated. Immunohistochemistry showed that Ndrg2 expression was reduced and that c-Myc was increased in colorectal carcinomas. In addition, Ndrg2 protein levels increased from poorly differentiated to well-differentiated carcinomas (p=0.005). Real-time polymerase chain reaction and Western blots demonstrated quantitatively that NDRG2 mRNA and protein levels were lower in colorectal carcinomas compared to the adjacent tissue and normal tissue from the same individual (p=3x10(-8)). Also, the NDRG2 expression level in adjacent carcinoma tissue was lower than that of normal tissue. However, the expression pattern of c-MYC was the inverse (p=5x10(-8)). Finally, we induced the differentiation of the colorectal carcinoma cell lines HT29, SW480 and SW620 and found that NDRG2 expression increased and that c-MYC expression declined with increasing differentiation. These novel data show a disparity in both the mRNA and protein expression levels of Ndrg2 and c-Myc between colorectal cancers and normal tissues. Taken together, NDRG2 may play a role during the differentiation of colorectal cancer cells, and the function of NDRG2 in the development of colorectal cancer should be further investigated.

The influence of survivin shRNA on the cell cycle and the invasion of SW480 cells of colorectal carcinoma

BackgroundThe objective was to understand the influence of Survivin plasmid with short hairpin RNA (shRNA) on the cell cycle, invasion, and the silencing effect of Survivin gene in the SW480 cell of colorectal carcinoma.MethodsA eukaryotic expression vector, PGCH1/Survivin shRNA, a segment sequence of Survivin as target, was created and transfected into colorectal carcinoma cell line SW480 by the non-lipid method. The influence on the Survivin protein was analyzed by Western blotting, while the cell cycle, cell apoptosis were analyzed by flow cytometry, and invasion of the cell was analyzed by Transwell's chamber method.ResultsAfter the transfection of PGCH1/Survivin shRNA, the expression of Survivin protein in SW480 cells was dramatically decreased by 60.68%, in which the cells were stopped at G2/M phase, even though no apoptosis was detected. The number of transmembranous cells of the experimental group, negative control group, and blank control group were 14.46 ± 2.11, 25.12 ± 8.37, and 25.86 ± 7.45, respectively (P <0.05).ConclusionSurvivin shRNA could significantly reduce the expression of Survivin protein and invasion of SW480 cells. Changes in cell cycle were observed, but no apoptosis was induced.

Identification of 5-fluorouracil response proteins in colorectal carcinoma cell line SW480 by two-dimensional electrophoresis and MALDI-TOF mass spectrometry

Colorectal cancer (CRC) is the second most prevalent cause of cancer-related deaths in the Western world. 5-Fluorouracil (5-FU) is a standard chemotherapeutic drug to treat CRC. However, the response rate is less than 20% and patients who have responded to 5-FU may become resistant. Therefore there is an urgent need to examine the 5-FU response proteins so that patients with no response to 5-FU can change to other treatment strategies promptly. In this study, the proteomic expression profile in a CRC cell line SW480 before and after 5-FU treatment was examined using 2-dimensional electrophoresis technology. Fourteen proteins with differential expression were identified using mass spectrometry and 7 of them were validated using immunocytochemical (ICC) staining. Protein identification indicated that cyclophilin A, cytokeratin 19 (CK19), cytokeratin 8 (CK8), ras-related nuclear protein, heat shock protein 27 (hsp27) and peroxiredoxin 6 (Prx 6) were upregulated whereas heat shock protein 60 (hsp60), cytokeratin 18 (CK18), cytokeratin 9 (CK9), carbamoylphosphate synthetase I, alpha-enolase, heat shock protein 70 (hsp70), nm23 and beta-actin were down-regulated. Seven of the 14 proteins detected were validated by ICC staining, which showed that the expression of hsp27, Prx 6 and hsp70 correlated with that from proteomics profiling. Our results suggest that hsp27, Prx 6 and hsp70 are potential 5-FU response proteins and they may represent potential targets for further evaluation in other 5-FU-sensitive and -resistant CRC cell lines.

Hyperthermia enhanced the killing effect of 5-fluorocytosine on human colon cancer cell line transfected with cytosine deaminase gene

To investigate whether hyperthermia can enhance the killing effect of 5- fluorocytosine (5- FC) on human colorectal carcinoma cell lines SW480 transfected with carcinoembryonic antigen (CEA) tissue- specific cytosine deaminase (CD) gene in vitro,and study its mechanism. Human colorectal carcinoma cell lines SW480 transfected with G1CEACDNa were cultured. The proliferated colonies were treated with the combined therapy of 5-FC and hyperthermia at a temperature of 43 degrees C for 30 min. After eight days, MTT was used to calculate the cellular survival rate,to analyze the killing effect of 5-FC combined with hyperthermia on SW480 cells transfected with CD gene. Flow cytometry was performed to analyze the cellular cycle and transmission electron microscope was used to observe the morphologic changes of SW480 cells after thermochemotherapy. Hyperthermia combined with 5-FC had an enhanced killing effect on SW480-CEACD cells than 5-FC alone (P< 0.05, t =2.403, n=9). Flow cytometry revealed that the proportion of S stage cell increased in the group treated with hyperthermia and 5- FC (P< 0.001, t =7.158, n=6). Transmission electron microscope showed apoptosis after thermo- chemotherapy. Hyperthermia can improve the anti- tumor effect of 5- FC on human colorectal carcinoma cell lines SW480 transfected with CD gene, and the cells were blocked at S stage of cellular cycle and apoptosis was induced following thermochemotherapy.

Antitumor effects and radiosensitization of cytosine deaminase and thymidine kinase fusion suicide gene on colorectal carcinoma cells.

To investigate the killing effect and radiosensitization of double suicide gene mediated by adenovirus on colorectal carcinoma cells. Colorectal carcinoma cell line SW480 was transfected with adenovirus expression vector containing cytosine deaminase (CD) and thymidine kinase (TK) fusion gene. The expression of CD-TK fusion gene was detected by reverse transcriptase-polymerase chain reaction. The toxic effect of ganciclovir (GCV) and 5-fluorocytosine (5-FC) on infected cells was determined by MTT assay. The radiosensitization of double suicide gene was evaluated by clonogenic assay. After prodrugs were used, the survival rate of colorectal carcinoma cells was markedly decreased. When GCV and 5-FC were used in combination, the cytotoxicity and bystander effect were markedly superior to a single prodrug (chi2 = 30.371, P<0.01). Both GCV and 5-FC could sensitize colorectal carcinoma cells to the toxic effect of radiation, and greater radiosensitization was achieved when both prodrug were used in combination. CD-TK double suicide gene can kill and radiosensitize colorectal carcinoma cells.

Identification of proteins of human colorectal carcinoma cell line SW480 by two-dimensional electrophoresis and MALDI-TOF mass spectrometry

To conduct the proteomic analysis of human colorectal carcinoma cell line, SW480 by using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption /ionization-time of flight mass spectrometry (MALDI-TOFMS). The total proteins of human colorectal carcinoma cell line, SW480 were separated with 2-DE by using immobilized pH gradient strips and visualized by staining with silver nitrate. The gel images were acquired by scanner and 2-DE analysis software, Image Master 2D Elite. Nineteen distinct protein spots were excised from gel randomly and digested in gel by TPCK-trypsin. Mass analysis of the tryptic digest peptides mixture was performed by using MALDI-TOF MS. Peptide mass fingerprints (PMFs) obtained by the MALDI-TOF analysis were used to search NCBI, SWISS-PROT and MSDB databases by using Mascot software. PMF maps of all spots were obtained by MALDI-TOF MS and thirteen proteins were preliminarily identified. The methods of analysis and identification of protein spots of tumor cells in 2-DE gel with silver staining by MALDI-TOF MS derived PMF have been established. Protein expression profile of SW480 has been obtained. It is demonstrated that a combination of proteomics and cell culture is a useful approach to comprehend the process of colon carcinogenesis.

Killing effect of CD/5-FC system on human colon cancer cell lines SW 480 and LoVo

AIM:To investigate the killing effect of carcinoembryonic antigen (CEA) and tissue-specific cytosine deaminase (CD)/5fluorocytosine (5-FC) system on human colorectal carcinoma cell lines LoVo and SW480 in vitro. METHODS:Recombinant retroviral vector G1CEACDNa was constructed,in which the CD gene was controlled under the CEA promoter, and retroviral vector pCD2 were introduced through liposome technique respectively to the human colorectal carcinoma cell lines LoVo and SW480. Expression of CEA was high and low in both the cell lines respectively. The cells were selectively cultured in G418. The proliferative colonies were treated with 5-FC. RESULTS:After the transfection, LoVo-CEACD cells and LoVo-CD cells were more sensitive to 5-FC than their parental cells (P <0.01, t =5.688, n =9; P <0.01, t =3.136, n =9), and SW480-CEACD cells and SW480-CD cells were more sensitive than their parental cells as well (P <0.01,t =3.437,n =9;P <0.01, t =3.516, n =9). Furthermore, the LoVo-CEACD cells were more sensitive to 5-FC than the LoVo-CD cells (P <0.05, t =2.183,n =9) while the SW480-CEACD cells were less sensitive than SW480-CD cells.The LoVo-CEACD cells displayed a higher anti-tumor effect than SW480-CEACD cells in vitro. The bystander effect in all cells transfected with CD gene were observed in this study. CONCLUSION:The CEA tissue-specific CD/5-FC system displays an obvious targeting anti-tumor effect on human colorectal carcinoma cell lines LoVo and SW480, but the killing effect on the LoVo-CEACD cells is higher than that on the SW480-CEACD cells in vitro. Li CJ, Ma QJ, Lai DN, Lu JG, Wang XJ, Wang Q, Pan BR, Wu YZ, Li JM. Killing effect of CD/5-FC system on human colon cancer cell lines SW 480 and LoVo. Shijie Huaren Xiaohua Zazhi 2003;11(5):535-539

Influence of cytokines, monoclonal antibodies and chemotherapeutic drugs on epithelial cell adhesion molecule (EpCAM) and LewisY antigen expression.

MoAbs against tumour-associated antigens (TAA) may be useful for the treatment of colorectal cancer. Since an increased expression of TAA may lead to enhanced antibody-dependent cellular cytotoxicity we examined whether the cytokines IL-2, IL-4, IL-6, IL-10, IL-12, interferon-alpha (IFN-alpha), IFN-gamma, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor and tumour necrosis factor-alpha can influence EpCAM and LewisY expression on the surface of the colorectal carcinoma cell lines HT29, LoVo and SW480. We found that only IFN-alpha increased significantly whereas IL-4 decreased both EpCAM and LewisY expression. IFN-gamma significantly increased LewisY expression only. When tumour cells were treated with MoAb, the LewisY-specific MoAb BR55-2 down-regulated LewisY antigen expression, whereas MoAb 17-1A, which binds to EpCAM, up-regulated this TAA after 3 days of culture. The cytokines IFN-alpha or IFN-gamma combined with MoAb 17-1A enhanced further slightly the expression of EpCAM. In additional experiments with chemotherapeutic drugs commonly used for the treatment of colorectal cancer, we found that 5-fluorouracil, mitomycin-C and oxaliplatin up-regulated EpCAM and LewisY antigen expression. Raltitrexed enhanced LewisY and down-regulated EpCAM expression, whereas CPT-11 had no influence at all. The highest expression for EpCAM on HT29 cells was achieved by the combination of IFN-alpha, 5-fluorouracil and MoAb 17-1A. Our results may be useful for defining combinations of biological and chemotherapeutic drugs for the treatment of colorectal cancer. Further trials should evaluate to what extent these combinations enhance antibody-dependent cellular cytotoxicity.

Expression of 15-lipoxygenase-1 is regulated by histone acetylation in human colorectal carcinoma.

15-Lipoxygenase-1 (15-LO-1) is expressed at higher levels in human colorectal tumors compared with normal tissue. 15-LO-1 is expressed in cultured human colorectal cells, but only after treatment with sodium butyrate (NaBT), which also stimulates apoptosis and cell differentiation. We examined the regulation of 15-LO-1 in human tissue and the colorectal carcinoma cell lines Caco-2 and SW-480 by treatment with histone deacetylase (HDAC) inhibitors: NaBT, trichostatin A (TSA) and HC toxin. Northern and western analysis showed that expression of 15-LO-1 was up-regulated by these HDAC inhibitors. Furthermore, HDAC inhibitors stimulated promoter activity of the 15-LO-1 gene approximately 12-to 21-fold using the -331/-23 region of the 15-LO-1 promoter, as measured with a luciferase-15-LO-1 promoter-reporter system, suggesting that 15-LO-1 is regulated at the transcriptional level by HDAC inhibitors. Histone proteins in colorectal cells were acetylated after treatment with HDAC inhibitors. Histone acetylation was also measured in human colorectal tissue and a correlation was observed between increased histone acetylation and 15-LO-1 expression. Thus, regulation of 15-LO-1 expression in colorectal tissues appears to occur by a novel and new mechanism associated with histone acetylation. Moreover, these results suggest that 15-LO-1 is a marker that reflects histone acetylation in colorectal carcinoma.

Immune reactions induced by interleukin-2 transfected colorectal cancer cells in vitro: predominant induction of lymphokine-activated killer cells.

One aim of the genetic modification of tumor cells is the generation of immunogenic variants that can be used for the induction of immune responses against tumors. We engineered the human colorectal carcinoma cell line SW480 by means of plasmid transfection to secrete interleukin (IL)-2. Transfection of SW480 cells resulted in stable IL-2 secretion at 5-30 ng/ml per 10(5) cells in 24 h and, unexpectedly, in CD54 expression on the cell surface. SW480 variants expressing IL-2 and CD54 were tested for their capacity to induce T lymphocyte activation in vitro in comparison to untransfected and CD54 transfected cells. The cytolytic effector function of a class I MHC restricted CD8+, peptide antigen specific T cell clone was augmented following expression of CD54. IL-2 secreting SW480 variants did not further increase antigen-dependent cytolysis. Primary activation of resting T lymphocytes was assessed following allogeneic stimulation. When compared with unmodified SW480 cells, CD54 expressing variants did not initiate T cell proliferation. In contrast, IL-2 secreting SW480 cells strongly promoted primary T cell proliferation. Similarly, exogenous IL-2 and SW480 cells induced T cell proliferation which was not only due to IL-2 but was dependent on tumor cells. However, following the initial wave of cell growth in response to IL-2 secreting SW480 cells T lymphocytes could not be restimulated with SW480 or IL-2 secreting variants and could not be further expanded. T cells initially activated by IL-2 secreting SW480 cells exhibited cytolytic activity towards SW480 cells. This reactivity, however, was transient and completely blocked by K562 cells, suggesting MHC-unrestricted, nonspecific cytotoxicity. We conclude that endogenous IL-2 secretion by the colorectal carcinoma cell line SW480 does not result in the activation of MHC restricted specific T lymphocytes but predominantly induces lymphokine-activated killer cells. Considering that tumor cell vaccines are aimed at inducing tumor-specific immune responses, our in vitro observation would rather argue against the in vivo application of such a tumor cell modification in colorectal cancer.