Colon Adenocarcinoma Cell Lines SW480 Research Articles

Boric acid suppresses cell proliferation by TNF signaling pathway mediated apoptosis in SW-480 human colon cancer line

Background/AimColon cancer is one of the most common cancers. Treatment success and survival rates are not high enough with current approaches. Therefore, there is a need to develop new agents and treatment methods. Boric acid is the most frequently observed form of boron. Some epidemiological data suggest that environmental exposure to boric acid reduces the incidence of prostate cancer in men, cervical and lung cancers in women. Experimental studies show, boric acid reduces cell proliferation and stimulates apoptosis in some prostate, melanoma, breast cancer cell lines. In this study, it was investigated whether boric acid could be a new candidate molecule that could be used in the treatment of colon cancer. Materials and methodsThe effects of boric acid on human colon adenocarcinoma cell line SW-480 were investigated with BrdU, TUNEL, Caspase-3, and AIF immunohistochemical studies in both 2D and 3D culture systems. In addition, a qRT-PCR study was carried out to determine the expression changes in key genes that take part in apoptosis. ResultsWe observed that boric acid suppresses cell proliferation and induces apoptosis both in 2D and 3D culture conditions. In addition, as a result of qRt-PCR studies, it was revealed that the observed apoptotic process was related to the TNF signaling pathway. ConclusionBoric acid can be considered as a potential anti-cancer agent candidate for colon cancer treatment. Data AvailabilityAll data generated or analyzed during this study are included in this published article.

TRAIL-expressing recombinant Lactococcus lactis induces apoptosis in human colon adenocarcinoma SW480 and HCT116 cells.

We investigated the ability of Lactococcus lactis, a species generally regarded as safe, to express tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) protein. The expressed protein was either cell wall anchored or secreted, and it was assessed whether this could induce apoptosis in human colon adenocarcinoma cell lines SW480 and HCT116. Constructs were designed to produce either secreted or cell wall-anchored forms of human TRAIL cloned into pNZ7021 expression vector. The expression by L. lactis was confirmed by Western blotting and immunofluorescence. Induction of cell death was evaluated by coculturing transformants producing either form of TRAIL protein with the two cell lines followed by MTT assay. Gene expression of apoptosis genes, Bax and Bcl2, was assessed by qPCR. The viability of SW480 and HCT116 cells treated with recombinant L. lactis was significantly reduced. A significant change was observed in the ratio of Bax/Bcl2 expression in HCT116 cells only following treatment with the supernatant of recombinant L. lactis containing secreted TRAIL. Recombinant L. lactis producing TRAIL protein can induce apoptosis in human colon adenocarcinoma cell lines SW480 and HCT116. The use of recombinant probiotics that produce anticancer compounds is a promising option for combating cancer cells.

Differential expression of ST6GAL1 in the tumor progression of colorectal cancer

Elevated expression of β-galactoside α2,6-sialyltranferase 1 (ST6GAL1) has been observed in colorectal cancer (CRC) and demonstrated to be important for its tumorigenesis. Here, we found that ST6GAL1 expression was significantly higher in non-metastatic tumors (stage I and II) than that in metastatic tumors (stage III and IV) using 62 pair-matched tumor/normal tissues. To elucidate the molecular mechanisms of how ST6GAL1 affected the CRC progression, we performed a global identification of the substrates of ST6GAL1 in the colon adenocarcinoma cell line SW480. A total of 318 membrane proteins were identified differentially affected by ST6GAL1 overexpression using metabolic labeling and proteomic analysis. Subsequent bioinformatic analysis revealed a list of potential substrates that might mediate the different functions of ST6GAL1 in CRC including cell movement, cell death and survival. Taken together, these results indicate a dynamic change in the expression of ST6GAL1 during the CRC progression and provide a list of sialylated proteins potentially relevant to the different functions of ST6GAL1 in CRC.

Abstract LB-059: Opposing roles for protein tyrosine kinase 6 (PTK6) in colon cancer

Abstract Background: Protein Tyrosine Kinase 6 (PTK6, also called BRK) is a non-receptor tyrosine kinase expressed in differentiated epithelial cells of the skin, gastrointestinal tract, and prostate. Disruption of the Ptk6 gene led to impaired intestinal differentiation and increased intestinal proliferation in mice. However, PTK6 also has a tumor-promoting role in the colon, as disruption of the Ptk6 gene impaired carcinogen-induced STAT3 activation and tumorigenesis in mice. PTK6 also promotes survival of human colon cancer cell lines following DNA damaging treatments including γ-irradiation and chemotherapeutic drugs via STAT3 activation. Results: We examined and manipulated colon cancer cell lines to understand these two seemingly contradictory roles for PTK6 in the colon. The human colon adenocarcinoma cell lines SW480 and SW620 represent a primary colon tumor and a metastasis from the same patient, respectively. PTK6 is highly expressed in SW480 cells and significantly reduced in SW620 cells. Active PTK6, phosphorylated on tyrosine residue 342, is not detectable in either cell line. Stable knockdown of PTK6 in SW480 cells results in an epithelial-to-mesenchymal transition (EMT), evidenced by decreased E-cadherin expression and increased ZEB-1 and Vimentin; as well as increased migration, wound-healing, and anchorage-independent growth of cells in culture. Additionally, knockdown of PTK6 led to increased in vivo tumorigenesis of xenograft cells in nude mice. Ectopic expression of wild-type PTK6 in SW620 cells drives the reverse process (MET), demonstrating a rescue of the EMT phenotype. When a constitutively active PTK6 construct is expressed in SW620 cells, it promotes activation of cytoplasmic oncogenic targets and downstream effectors, STAT3, FAK, BCAR1 and ERK5. Analysis of patient tissues demonstrates a downregulation of mRNA and protein levels, as well as a change in protein localization in tumor versus normal tissues; while PTK6 is detectable in the nuclei of differentiating normal cells it is excluded from nuclei in colon tumors. Conclusions: Our studies indicate that PTK6 promotes the epithelial phenotype to antagonize the EMT in a kinase-independent manner and a kinase switch activates PTK6 to promote oncogenic signaling. PTK6 protein localization may function to regulate its specific role. Further examination of the complex role of this kinase could allow for a more nuanced understanding of colon cancer initiation and progression for the development of novel treatments. Citation Format: Priya S. Mathur, Jessica J. Gierut, Rosa M. Xicola, Xavier Llor, Angela L. Tyner. Opposing roles for protein tyrosine kinase 6 (PTK6) in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-059. doi:10.1158/1538-7445.AM2015-LB-059

Role of paxillin in colorectal carcinoma and its relationship to clinicopathological features

Background Colorectal carcinoma is one of the most common malignant tumors. Despite advances in therapy, mortality is still very high. The aim of this study was to evaluate the expression of paxillin in the human colon adenocarcinoma cell line SW480 and its role in cell cycle and apoptosis. We also investigated the expression of paxillin in colorectal carcinoma tissues and its relationship to clinicopathological features and survival. Methods Paxillin short hairpin RNA (shRNA) was constructed and transfected into the colon adenocarcinoma cell line SW480. The influence of paxillin shRNA on the cell cycle and cell apoptosis was analyzed by flow cytometry. Immunohistochemistry staining was used to assess the expression of paxillin and its association with the expression of carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9, p53 and Bcl-2 in 102 patients with primary colorectal carcinoma. Western blotting was also used to investigate the expression of paxillin. Medical records were reviewed and a clinicopathological analysis was performed. Results In vitro, the percentage of cells in S phase was (45.23±1.05)%, (43.53±1.23)%, and (36.13±0.57)% in the blank control group, negative control group, and paxillin shRNA group respectively. It was significantly decreased in the paxillin shRNA group (P=0.000). The early apoptosis index of the paxillin shRNA group (17.2±1.18%) was significantly increased compared to the control shRNA group ((13.17±1.15)%, P=0.013). Paxillin was positive in 71 (69.6%) patients, and it was found to be overexpressed in tumor tissues compared with normal adjacent tissues. Paxillin positive rate was higher in patients who are less than 50-years old (100.0% vs. 65.6%, P=0.016). Paxillin expression was associated with a high histologic grade of carcinoma (81.4% vs. 61.0%, P=0.031), a high rate of regional lymph node metastasis (22.5% vs. 13.0%, P=0.031), mesenteric artery lymph node metastasis (100.0% vs. 64.8%, P=0.008), distant metastasis (94.1% vs. 64.7%, P=0.016) and a high Tumor Node Metastasis (TNM) stage (94.1%, 73.2%, 60.0%, and 50%, P=0.030). Multivariate analyses revealed that recurrence was associated with the rate of regional lymph node metastasis (P=0.001) and paxillin expression (P=0.024). Multivariate analysis indicated that the overall survival is related to the TNM stage (P=0.000). Conclusions In vitro, paxillin may promote cell proliferation and inhibit apoptosis in SW480 cells. Paxillin may be a potential metastasis predictor, and an independent prognosis factor of recurrence. It may also be related to poor patient outcomes, but was not an independent predictor of survival.

Association of Nox1 and Vinculin With Colon Cancer Progression

Nox1 mRNA, protein, and activities were compared in the paired primary and metastatic colon adenocarcinoma cell lines SW480 and SW620, and in normal colon tissues and colon cancer tissues. Our results demonstrated that Nox1 levels were higher in the primary SW480 cells than that in metastatic SW620 cells and were not associated with colon cancer progression. We further discovered that vinculin protein level in SW620 was much higher than that in SW480 cells, whereas E-cadherin was lower. We conclude that vinculin and E-cadherin, but not Nox1, may serve as biomarkers for colon cancer progression.

A Functional Variant of IC53 Correlates with the Late Onset of Colorectal Cancer

The IC53 gene was reported to be upregulated in the colon adenocarcinoma cell line SW480. Here, we show that the expression level of IC53 is positively correlated with the grade and depth of invasion in adenocarcinoma of the colon. Injection of IC53 stably transfected HCT-116 cells into athymic nude mice promoted tumor growth. Furthermore, overexpression of IC53 increased cell invasive growth, which could be dramatically prevented by knocking down IC53 with siRNA. The effects of IC53 on cell-invasive growth were mediated by upregulation of integrins, activation of phosphatidylinositol 3-kinase and phosphorylation of Akt. A single-nucleotide polymorphism rs2737 in the IC53 gene created a potential microRNA379 target site, and microRNA379 expression inhibited IC53 translation. Among 222 patients with colorectal cancer, the C/C rs2737 genotype was associated with late onset of colorectal cancer (median age 63.0 versus 55.3 years, P = 0.003). The frequency of the C/C rs2737 genotype was much lower in patients who developed colorectal cancer below the age of 45 years than in individuals over age 45 years (10.8% versus 26.6%, P = 0.039). These data indicated that IC53 is a positive mediator for colon cancer progression, and IC53-rs2737 may serve as protection from the onset of colorectal cancer.

ShRNA-mediated down-regulation of paxillin reduces cell invasion in human colon adenocarcinoma cell line SW480

shRNA-mediated down-regulation of paxillin reduces cell invasion in human colon adenocarcinoma cell line SW480

Two Novel Monoclonal Antibodies Against Human CD133-2: Distinct Epitopes and Agonist Activity to Enhance Growth of CD133 Expression CellsIn Vitro

Human AC133 antigen, also called CD133, is a unique transmembrane glycoprotein encoded by the PROM1 gene. It was initially suggested as a cell surface marker for hematopoietic stem/progenitor cells and has also been identified recently as a cancer stem cell marker in brain, colorectal, and prostate cancers. AC133 has two isoforms, one is AC133-1 and the other is AC133-2. Whether the two isoforms are functionally redundant or serve distinct functions remains unclear. In order to further explore the physiological and pathological functions of CD133-2, we generated two mouse antihuman CD133-2 monoclonal antibodies (clones 6B3 and 9G4). Then, we carefully characterized the biological functions of these monoclonal antibodies (MAbs) and showed that 6B3 and 9G4 bound to two epitopes of CD133, which are different from that recognized by the commercially available anti-CD133 MAb (AC141). These two MAbs could be used in cell immunostaining, Western blot, and immunohistochemical staining. We have further shown that MAb 6B3 enhances the growth of human myelogenous leukemic cell line U937 and human colon adenocarcinoma cell line SW480, suggesting a functional role of CD133 in these cells. Taken together as a useful tool, these two antibodies might be of great value for further exploration of the expression and function of CD133.

Abstract: P673 EFFECT OF IL-17 ON INVASION OF HUMAN COLON CANCER CELLS IN VITRO

Elevated expression of β-galactoside α2,6-sialyltranferase 1 (ST6GAL1) has been observed in colorectal cancer (CRC) and demonstrated to be important for its tumorigenesis. Here, we found that ST6GAL1 expression was significantly higher in non-metastatic tumors (stage I and II) than that in metastatic tumors (stage III and IV) using 62 pair-matched tumor/normal tissues. To elucidate the molecular mechanisms of how ST6GAL1 affected the CRC progression, we performed a global identification of the substrates of ST6GAL1 in the colon adenocarcinoma cell line SW480. A total of 318 membrane proteins were identified differentially affected by ST6GAL1 overexpression using metabolic labeling and proteomic analysis. Subsequent bioinformatic analysis revealed a list of potential substrates that might mediate the different functions of ST6GAL1 in CRC including cell movement, cell death and survival. Taken together, these results indicate a dynamic change in the expression of ST6GAL1 during the CRC progression and provide a list of sialylated proteins potentially relevant to the different functions of ST6GAL1 in CRC.

Matrix Metalloproteinases 9 and 10 Inhibit Protein Kinase C–Potentiated, p53-Mediated Apoptosis

p53, a major sensor of DNA damage, is a transcription factor that, depending on its phosphorylation status, regulates the cell cycle, DNA repair, or apoptosis. The protein kinase C (PKC) family of isozymes is also implicated in cell cycle and programmed cell death (PCD) control and has recently been shown to influence p53 function. Using three human colon adenocarcinoma cell lines SW480, EB-1, and HCT116 that either lack p53 function and were engineered to express inducible wild-type p53 (wt p53), or that constitutively express wt p53, we show that phorbol ester-mediated PKC activation potentiates p53-induced PCD. Despite the effectiveness of PKC/p53 synergy in inducing SW480 tumor cell death, however, a fraction of the cells invariably survive. To address the putative mechanisms that underlie resistance to PKC/p53-induced cell death, we generated a phorbol 12-myristate 13-acetate/p53-resistant SW480 subline and compared the gene expression profile of resistant and parental cells by DNA microarray analysis. The results of these experiments show that PKC/p53-resistant cells express a higher level of several matrix metalloproteinases (MMP), including MMP-9, MMP-10, and MMP-12, and corresponding real-time PCR assays indicate that p53 is a negative regulator of MMP-9 gene expression. Using MMP inhibitors and MMP-specific small interfering RNA, we show that MMP function confers protection from PKC/p53-induced apoptosis and identify the protective MMPs as MMP-9 and MMP-10. Taken together, these observations provide evidence that MMPs are implicated in tumor cell resistance to the synergistic proapoptotic effect of PKC and p53.

Hsp72 Inhibits Fas-mediated Apoptosis Upstream of the Mitochondria in Type II Cells

Heat shock protein 72 (Hsp72) inhibits apoptosis induced by some stresses that trigger the intrinsic apoptosis pathway. However, with the exception of TNFalpha-induced apoptosis, a role for Hsp72 in modulating the extrinsic pathway of apoptosis has not been clearly established. In this study, it was demonstrated that Hsp72 could inhibit Fas-mediated apoptosis of type II CCRF-CEM cells, but not type I SW480 or CH1 cells. Similar results were obtained when Fas ligand or an agonistic Fas antibody initiated the Fas apoptosis pathway. In CCRF-CEM cells, Hsp72 inhibited mitochondrial membrane depolarization and cytochrome c release but did not alter surface Fas expression or processing of caspase-8 and Bid, indicating that Hsp72 acts upstream of the mitochondria to inhibit Fas-mediated apoptosis. Thus, the ability of Hsp72 to inhibit Fas-mediated apoptosis is limited to type II cells where involvement of the intrinsic pathway is required for efficient effector caspase activation.

Patupilone Acts as Radiosensitizing Agent in Multidrug-Resistant Cancer Cells In vitro and In vivo

Interference with microtubule function is a promising antitumoral concept. Paclitaxel is a clinically validated tubulin-targeting agent; however, treatment with paclitaxel is often limited by taxane-related toxicities and is ineffective in tumors with multidrug-resistant cells. Patupilone (EPO906, epothilone B) is a novel non-taxane-related microtubule-stabilizing natural compound that retains full activity in multidrug-resistant tumors and is clinically less toxic than paclitaxel. Here we have investigated the effect of combined treatment with ionizing radiation and patupilone or paclitaxel in the P-glycoprotein-overexpressing, p53-mutated human colon adenocarcinoma cell line SW480 and in murine, genetically defined E1A/ras-transformed paclitaxel-sensitive embryo fibroblasts. Patupilone and paclitaxel alone and in combination with ionizing radiation reduced the proliferative activity of the E1A/ras-transformed cell line with similar potency in the sub and low nanomolar range. SW480 cells were only sensitive to patupilone, and combined treatment with low-dose patupilone (0.1 nmol/L) followed by clinically relevant doses of ionizing radiation (2 and 5 Gy) resulted in a supra-additive cytotoxic effect. Inhibition of the drug efflux protein P-glycoprotein with verapamil resensitized SW480 cells to treatment with low doses of paclitaxel alone and in combination with IR. In tumor xenografts derived from SW480 cells a minimal treatment regimen with patupilone and fractionated irradiation (1 x 2 mg/kg plus 4 x 3 Gy) resulted in an at least additive tumor response with extended tumor growth arrest. Analysis by flow cytometry in vitro revealed an apoptosis- and G(2)-M-independent mode of radiosensitization by patupilone. Interestingly though, a transient accumulation of cells in S phase was observed on combined treatment.Overall, patupilone might be a promising alternative in paclitaxel-resistant, P-glycoprotein-overexpressing tumors for a combined treatment regimen using ionizing radiation and a microtubule inhibitor.

Carcinoembryonic antigen (CEA) presentation and specific T cell-priming by human dendritic cells transfected with CEA-mRNA

The feasibility of dendritic cells (DC) for cancer immunotherapy after transfection by electroporation with mRNA encoding the human carcinoembryonic antigen (CEA) was investigated. Both, total RNA from the CEA+ colon cancer cell line SW480 and mRNA transcribed in vitro from cDNA3.1-plasmids (pcDNA3.1+/-HisC) with a CEA-insert (ivt-CEA-mRNA, ivt-CEA/HisC-mRNA) were used. Labelled ivt-CEA-mRNA was detectable in DC by light and electron microscopy and by fluorescence-activated cell-sorting (FACS) even 15 min after electroporation. Four hours after transfection with ivt-CEA/HisC-mRNA, we detected specific expression of CEA and the histidine-tag by immunofluorescence microscopy and by FACS. CEA-specific T lymphocytes were successfully primed by transfected DC and were able to lyse CEA-expressing target cells, even from the CEA-expressing human colon adenocarcinoma cell line SW480. Thus, DC transfected by electroporation with CEA-mRNA are valuable tools for the immunotherapy of CEA+ tumour entities.