SW480 Human Colon Adenocarcinoma Cells Research Articles

Identification of phenolic compounds from inflorescences of non-psychoactive Cannabis sativa L. by UHPLC-HRMS and in vitro assessment of the antiproliferative activity against colorectal cancer

Phenolic compounds from Cannabis sativa L. (Cannabaceae family), in particular cannflavins, are known to possess several biological properties. However, their antiproliferative activity, being of great interest from a medicinal chemistry point of view, has not been deeply investigated so far in the literature. In the light of this, the aim of this study was to obtain an enriched fraction of polyphenols (namely PEF) from inflorescences of a non-psychoactive C. sativa (hemp) variety and to evaluate its antiproliferative activity against cancer cells, capitalizing on a new and selective extraction method for hemp polyphenols, followed by preparative flash column chromatography. Untargeted metabolomics, using a new method based on ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS), was applied here for the first time to fully characterize PEF. Then, the main phenolic compounds were quantified by HPLC-UV. The antiproliferative activity of PEF and of the isolated compounds was assessed in vitro for the first time against Caco-2 and SW480 human colon adenocarcinoma cell lines providing promising IC50 values, in comparison with the reference drug used in therapy for this cancer type. Based on these results, PEF can be considered as a new highly potential therapeutic product to be further investigated against colorectal cancer, thanks to the possible synergistic interaction of its compounds.

Chemopreventive Effect on Human Colon Adenocarcinoma Cells of Styrylquinolines: Synthesis, Cytotoxicity, Proapoptotic Effect and Molecular Docking Analysis.

Seven styrylquinolines were synthesized in this study. Two of these styrylquinolines are new and were elucidated by spectroscopic analysis. The chemopreventive potential of these compounds was evaluated against SW480 human colon adenocarcinoma cells, its metastatic derivative SW620, and normal cells (HaCaT). According to the results, compounds 3a and 3d showed antiproliferative activity in SW480 and SW620 cells, but their effect seemed to be caused by different mechanisms of action. Compound 3a induced apoptosis independent of ROS production, as evidenced by increased levels of caspase 3, and had an immunomodulatory effect, positively regulating the production of different immunological markers in malignant cell lines. In contrast, compound 3d generated a pro-oxidant response and inhibited the growth of cancer cells, probably by another type of cell death other than apoptosis. Molecular docking studies indicated that the most active compound, 3a, could efficiently bind to the proapoptotic human caspases-3 protein, a result that could provide valuable information on the biochemical mechanism for the in vitro cytotoxic response of this compound in SW620 colon carcinoma cell lines. The obtained results suggest that these compounds have chemopreventive potential against CRC, but more studies should be carried out to elucidate the molecular mechanisms of action of each of them in depth.

Identification of a novel colon adenocarcinoma cell targeting peptide using phage display library biopanning.

Phage display is well recognized as a promising high-throughput screening tool for the discovery of novel cancer-targeting peptides. Here, we screened a phage display library of 7-mer random peptides through in vitro biopanning to isolate peptide ligands binding to SW480 human colon adenocarcinoma cells. Three rounds of negative and positive selection caused a remarkable enrichment of colon cancer cell-binding phage clones with a significant enhancement of phage recovery efficiency (about 157-fold). A number of phage clones were picked out from the eluted phages of last selection round and sequenced. According to the results of cell binding assay and phage cell-based ELISA, one of the isolated peptides denoted as CCBP1 (with the sequence HAMRAQP) was indicated to have the highest binding efficiency, selectivity, and specificity toward colon cancer cells with no significant binding to control cells. Peptide competitive inhibition assay revealed that binding of the phage-displayed CCBP1 is competitively inhibited by the same free peptide, suggesting that CCBP1 specific binding to the target cell is independent of the phage context. Taken together, our findings provide support for the notion that CCBP1 binds specifically to colon cancer cells and might be a potential lead candidate for targeted delivery of imaging agents or therapeutic genes/drugs to colon tumors.

New Hybrids Based on Curcumin and Resveratrol: Synthesis, Cytotoxicity and Antiproliferative Activity against Colorectal Cancer Cells.

We synthesized twelve hybrids based on curcumin and resveratrol, and their structures were elucidated by spectroscopic analysis. The chemopreventive potential of these compounds was evaluated against SW480 human colon adenocarcinoma cells, its metastatic derivative SW620, along with the non-malignant CHO-K1 cell line. Among the tested compounds, hybrids 3e and 3i (for SW480) and 3a, 3e and 3k (for SW620) displayed the best cytotoxic activity with IC50 values ranging from 11.52 ± 2.78 to 29.33 ± 4.73 µM for both cell lines, with selectivity indices (SI) higher than 1, after 48 h of treatment. Selectivity indices were even higher than those reported for the reference drug, 5-fluorouracil (SI = 0.96), the starting compound resveratrol (SI = 0.45) and the equimolar mixture of curcumin plus resveratrol (SI = 0.77). The previous hybrids showed good antiproliferative activity.

Promising Hybrids Derived from S-Allylcysteine and NSAIDs Fragments against Colorectal Cancer: Synthesis, In-vitro Evaluation, Drug-Likeness and In-silico ADME/tox Studies.

We synthesized twelve hybrids, S-allyl Cysteine methyl, ethyl and propyl ester-based non-steroidal anti-inflammatory drugs and their structures were elucidated by spectroscopic analysis. The chemopreventive potential of all compounds was evaluated against SW480 human colon adenocarcinoma cells and the non-malignant CHO-K1 cell line. Among the tested compounds, hybrids 10b-c, 11b and 12b displayed the best anticancer activity with IC50 values between 0.131-0.183 mM and selectivity indices higher than 1 after 48 h of treatment. Selectivity indices were comparable to those reported for the reference drug, 5-fluorouracil (SI > 1). The SAR analysis showed that compounds with two carbon atom alkylic chains displayed the best activity (10b, 11b and 12b). Modeling studies including drug-likeness, bioactivity score and ADME/tox studies using online tools like molinspiration and Osiris suggested that these designed hybrids have a good pharmacological profile and can be considered as promising scaffolds for further studies in the search for new therapeutic alternatives to treat colorectal cancer.

Styrylcoumarin 7-SC2 induces apoptosis in SW480 human colon adenocarcinoma cells and inhibits azoxymethane-induced aberrant crypt foci formation in BALB/c mice

In vivo chemopreventive effects associated with hybrid molecules against colon carcinogenesis remain poorly studied. In a previous study, we showed that styrylcoumarin hybrids 3-SC1, 7-SC2 (2,7-(4-hydroxy-3,5-dimethoxystyryl)-coumarin), and 7-SC3 decrease cell viability of SW480 in a time- and concentration-dependent manner (IC50-SW480/48 h = 6.92; 1.01 and 5.33 µM, respectively) with high selectivity indices after 48 h of treatment (>400; 67.8 and 7.2, respectively). The present study investigates the mechanisms of these three styrylcoumarins to induce cell death, using an in vitro model of colon adenocarcinoma cells (SW480); besides, it evaluates anticarcinogenic properties in vivo for the most active molecule. According to the results, none of the hybrids exhibited significant changes in cell cycle distribution of SW480 cells with respect to control group (G0/G1 = 85.5%, S = 7.2%, and G2/M 7.3%), which indicates that these do not have a cytostatic effect on this cell line. Besides, they did not cause mitochondrial depolarization, suggesting an alternative source for the production of reactive oxygen species (ROS). Among the evaluated compounds, the most active molecule 7-SC2 induced a greater production of ROS in comparison with the control (p < 0.05) together with a significant increase in the expression of p53, caspase-3, and a significant reduction in the production of interleukin-6 of SW480 cells. When colon carcinogenesis was induced in Balb/c mice by intraperitoneal injections of azoxymethane, a significant reduction (p < 0.05) in the number of preneoplastic lesions of mice treated with styrylcoumarin hybrid 7-SC2 was observed with regard to the control group. In addition, no side effects were associated with the administration of the compound. All these in vitro results and the effective reduction of preneoplastic lesions in vivo suggest that styrylcoumarin 7-SC2 induces apoptosis in primary tumor cells and implies the potential ability at the early post-initiation phases of colon carcinogenesis. Moreover, hybrid 7-SC2 was docked to the three-dimensional structures of different apoptotic proteins and inflammatory cytokines, showing high binding affinities (ranging from −10.0 to −7.2 kcal/mol). Good correlation between calculated binding energies and experimental results was obtained. According to in silico ADME (absorption, distribution, metabolism, and excretion) studies of the 7-SC2, this novel compound has suitable drug-like properties, making it a potentially promising agent for therapy against colon cancer.

Synthesis and in-vitro Evaluation of S-allyl Cysteine Ester - Caffeic Acid Amide Hybrids as Potential Anticancer Agents.

We have synthesized a series of S-allyl cysteine ester-caffeic acid amide hybrids and evaluated them in order to determine their possible anticancer activity and selectivity in colorectal cancer, which is still one of the leading causes of morbidity and mortality worldwide. All compounds were tested against SW480 human colon adenocarcinoma cells and the non-malignant CHO-K1 cell line. Among the tested compounds, hybrids 6e, 9a, 9b, 9c, and 9e exhibited the highest effect on viability (IC50 SW480-48h= 0.18, 0.12, 0.12, 0.11, and 0.12 mM, respectively) and selectivity (SI = 10.3, 1.5, >83.33, >90.91 and >83.33, respectively) in a time- and concentration-dependent manner. Besides, our results were even better as regards lead compounds (S-allyl cysteine and caffeic acid) and the standard drug (5-FU). Additionally, these five compounds induced mitochondrial depolarization that could be related with an apoptotic process. Moreover, hybrids 6e, 9a, and 9e induced cell cycle arrest in G2/M phase, and compound 9c in S- phase, which suggests that these hybrid compounds could have also a cytostatic effect in SW480 cell line. The SAR analysis showed that hydroxyl groups increased the activity. Besides, there was not a clear relationship between the antitumor properties and the length of the alkyl chain. Since hybrid compounds were much more selective than the conventional drug (5-FU), this makes them promising candidates for further studies against colorectal cancer.

Integrins are not essential for entry of coxsackievirus A9 into SW480 human colon adenocarcinoma cells.

BackgroundCoxsackievirus A9 (CV-A9) is a pathogenic enterovirus type within the family Picornaviridae. CV-A9 infects A549 human epithelial lung carcinoma cells by attaching to the αVβ6 integrin receptor through a highly conserved Arg-Gly-Asp (RGD) motif, which is located at the exposed carboxy-terminus of the capsid protein VP1 detected in all studied clinical isolates. However, genetically-modified CV-A9 that lacks the RGD motif (CV-A9-RGDdel) has been shown to be infectious in some cell lines but not in A549, suggesting that RGD-mediated integrin binding is not always essential for efficient entry of CV-A9.MethodsTwo cell lines, A549 and SW480, were used in the study. SW480 was the study object for the integrin-independent entry and A549 was used as the control for integrin-dependent entry. Receptor levels were quantitated by cell sorting and quantitative PCR. Antibody blocking assay and siRNA silencing of receptor-encoding genes were used to block virus infection. Peptide phage display library was used to identify peptide binders to CV-A9. Immunofluorescence and confocal microscopy were used to visualize the virus infection in the cells.ResultsWe investigated the receptor use and early stages of CV-A9 internalization to SW480 human epithelial colon adenocarcinoma cells. Contrary to A549 infection, we showed that both CV-A9 and CV-A9-RGDdel internalized into SW480 cells and that function-blocking anti-αV integrin antibodies had no effect on the binding and entry of CV-A9. Whereas siRNA silencing of β6 integrin subunit had no influence on virus infection in SW480, silencing of β2-microglobulin (β2M) inhibited the virus infection in both cell lines. By using a peptide phage display screening, the virus-binding peptide identical to the N-terminal sequence of HSPA5 protein was identified and shown to block the virus infection in both A549 and SW480 cell lines. HSPA5 was also found to co-localize with CV-A9 at the SW480 cell periphery during the early stages of infection by confocal microscopy.ConclusionsThe data suggest that while αVβ6 integrin is essential for CV-A9 in A549 cell line, it is not required in SW480 cell line in which β2M and HSPA5 alone are sufficient for CV-A9 infection. This suggests that the choice of CV-A9 receptor(s) is dependent on the tissue/cellular environment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0619-y) contains supplementary material, which is available to authorized users.

Methyl nomilinate from citrus can modulate cell cycle regulators to induce cytotoxicity in human colon cancer (SW480) cells in vitro

Limonoids are triterpenoids found in citrus and possess cancer preventive properties in in vitro and in vivo assays. Although several mechanisms for the chemopreventive properties of limonoids have been postulated, the specific mechanisms involved in the anti-cancer effects have not been explored. In the present study, limonoids, including methyl nomilinate, isoobacunoic acid, isolimonexic acid (ILNA), and limonexic acid (LNA), were purified, identified by LC–MS and NMR spectral data and evaluated for their biological effects on SW480 human colon adenocarcinoma cells. Methyl nomilinate was the most potent inhibitor of cell metabolic activity in MTT and EdU incorporation assays. These limonoids did not affect apoptotic markers such as caspase-3 and PARP, but methyl nomilinate treatment resulted in significant induction of G0/G1 cell cycle arrest. Furthermore, methyl nomilinate suppressed CDK4/6 and cyclin D3 and the expression of CDK inhibitors. Taken together, the results suggest inhibition of cell proliferation by methyl nomilinate occurs due to G1 cell cycle arrest, indicating that methyl nomilinate has potential as a chemopreventive agent.

Silencing of the Insulin Receptor Isoform A Favors Formation of Type 1 Insulin-Like Growth Factor Receptor (IGF-IR) Homodimers and Enhances Ligand-Induced IGF-IR Activation and Viability of Human Colon Carcinoma Cells

Insulin receptor (IR) overexpression is common in cancers, with expression of the A isoform (IR-A, exon 11-) predominating over the B isoform. The IR-A signals a proliferative, antiapoptotic response to IGF-II, which itself can be secreted by tumors to establish an autocrine proliferative loop. Therefore, IGF-II signaling via the IR-A could mediate resistance to type 1 IGF receptor (IGF-IR) inhibitory drugs that are currently in development. This study addressed the role of the IR-A, using a small interfering RNA-based approach in SW480 human colon adenocarcinoma cells that coexpress the IGF-IR. Clonogenic survival was inhibited by depletion of the IGF-IR but not the IR-A, and dual receptor depletion had no greater effect than IGF-IR knockdown alone, suggesting that the IR-A could not compensate for IGF-IR loss. IGF-IR knockdown also resulted in a decrease in viability, whereas IR-A depletion resulted in increased viability. Consistent with this, upon IR-A depletion, we found a concomitant enhancement of IGF-IR activation by IGF-I and IGF-II, reduced formation of IGF-IR:IR-A hybrid receptors and increased IGF-IR homodimer formation. Together, these results suggest that IGF bioactivity is mediated more effectively by the IGF-IR than by the IR-A or receptor hybrids and that signaling via the IGF-IR is dominant to the IR-A in colon cancer cells that express both receptors.

Cysteine 62 of Bax Is Critical for Its Conformational Activation and Its Proapoptotic Activity in Response to H2O2-induced Apoptosis

Bax is activated and translocated onto mitochondria to mediate cytochrome c release and apoptosis. The molecular mechanisms of Bax activation during apoptosis remain a subject of debate. We addressed the question of whether reactive oxygen species could directly activate Bax for its subsequent translocation and apoptosis. Using the SW480 human colon adenocarcinoma cell line stably expressing Bax fused to GFP, we showed that H2O2 induces Bax conformational change, mitochondrial translocation, and subsequent oligomerization at mitochondria. We found that H2O2-induced Bax activation is dependent on the conserved cysteine residue 62 of Bax. Mutation of cysteine 62, but not cysteine 126, to serine or alanine abolished its activation by H2O2 but not other death stimuli, both in SW480 and Bax-deficient HCT116 cells, whereas wild type Bax sensitizes these cells to apoptosis. Cysteines of Bax could chemically react with H2O2. Mutation of Bax BH3 domain in the presence of cysteine 62 also abolished Bax proapoptotic activity. We conclude that reactive oxygen species could be a direct signal for Bax activation by reacting with cysteine residues. Our results identify a critical role of cysteine 62 in oxidative stress-induced Bax activation and subsequent apoptosis.

3: Sp1 regulates osteopontin expression in SW480 human colon adenocarcinoma cells

Introduction: Osteopontin (OPN), a ligand for alpha-v-beta-3 integrin and CD44 receptors, mediates metastatic behavior in a variety of cancers. However, the molecular mechanisms that regulate OPN gene expression in human colorectal cancer are unknown. Using SW480 human colon adenocarcinoma cells, we hypothesized that cis- and trans-transcriptional mechanisms determine OPN expression. Methods: SW480 human colon adenocarcinoma cells constitutively express OPN. Transient transfection and clonal deletion analysis of human OPN promoter (full-length 2.1 kb)-luciferase constructs identified cis-regulatory regions.

Aldose Reductase Regulates Growth Factor-Induced Cyclooxygenase-2 Expression and Prostaglandin E2 Production in Human Colon Cancer Cells

Inhibition of prostaglandin E(2) (PGE(2)) and cyclooxygenase (COX)-2 by nonsteroidal anti-inflammatory drugs reduces the progression of colon cancer. Inhibition of aldose reductase (AR; EC. 1.1.1.21.) by sorbinil or by antisense ablation prevented fibroblast growth factor-induced and platelet-derived growth factor-induced up-regulation of PGE(2) synthesis in human colon cancer cells, Caco-2. AR besides reducing aldo-sugars efficiently reduces toxic lipid aldehydes and their conjugates with glutathione. Inhibition of AR prevented growth factor-induced COX-2 activity, protein, and mRNA and significantly decreased activation of nuclear factor-kappaB and protein kinase C (PKC) and phosphorylation of PKC-beta2 as well as progression of Caco-2 cell growth but had no effect on COX-1 activity. Cell cycle analysis suggests that inhibition of AR prevents growth factor-induced proliferation of Caco-2 cells at S phase. Treatment of Caco-2 cells with the most abundant and toxic lipid aldehyde 4-hydroxy-trans-2-nonenal (HNE) or its glutathione-conjugate [glutathionyl-HNE (GS-HNE)] or AR-catalyzed product of GS-HNE, glutathionyl-1,4-dihydroxynonane (GS-DHN), resulted in increased COX-2 expression and PGE(2) production. Inhibition of AR prevented HNE- or GS-HNE-induced but not GS-DHN-induced up-regulation of COX-2 and PGE(2). More importantly, in vivo studies showed that administration of AR-small interfering RNA (siRNA), but not control siRNA, to nude mice bearing SW480 human colon adenocarcinoma cells completely arrested tumor progression. Collectively, these observations suggest that AR is an obligatory mediator of growth factor-induced up-regulation of COX-2, PGE(2), and growth of Caco-2 cells, indicating that inhibition of AR may be a novel therapeutic approach in preventing the progression of colon cancer.

The effects of quercetin on SW480 human colon carcinoma cells: a proteomic study

BackgroundHigh fruit and vegetable intake is known to reduce the risk of colon cancer. To improve understanding of this phenomenon the action of different phytochemicals on colon cells has been examined. One such compound is quercetin that belongs to the group known as flavonoids. The purpose of this study was to determine the influence of quercetin on the proteome of the SW480 human colon adenocarcinoma cell line, specifically to identify proteins that could be the molecular targets of quercetin in its amelioration of the progression of colon cancer. To this end, two-dimensional gel electrophoresis and mass spectrometry were used to identify proteins that underwent a change in expression following treatment of the cells with 20 μM quercetin. This could elucidate how quercetin may reduce the progression of colon cancer.ResultsQuercetin treatment of the SW480 human colon cancer cells was found to result in the decreased expression of three proteins and the increased expression of one protein. The identified proteins with decreased expression were type II cytoskeletal 8 keratin and NADH dehydrogenase Fe-S protein 3. The other protein with decreased expression was not identified. The protein with increased expression belonged to the annexin family.ConclusionSeveral proteins were determined to have altered expression following treatment with quercetin. Such changes in the levels of these particular proteins could underlie the chemo-protective action of quercetin towards colon cancer.

Deficient Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Death Receptor Transport to the Cell Surface in Human Colon Cancer Cells Selected for Resistance to TRAIL-induced Apoptosis

Many tumor cell types are sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Incubation of TRAIL-sensitive cells with TRAIL invariably leads to resistant survivors even when high doses of TRAIL are used. Because the emergence of resistance to apoptosis is a major concern in successful treatment of cancer, and TRAIL survivors may contribute to therapeutic failure, we investigated potential resistance mechanisms. We selected TRAIL-resistant SW480 human colon adenocarcinoma cells by repeatedly treating them with high and/or low doses of TRAIL. The resulting TRAIL-resistant clones were not cross-resistant to Fas or paclitaxel. Expression of modulators of apoptosis was not changed in the resistant cells, including TRAIL receptors, cFLIP, Bax, Bid, or IAP proteins. Surprisingly, we found that DISC formation was deficient in multiple selected TRAIL-resistant clones. DR4 was not recruited to the DISC upon TRAIL treatment, and caspase-8 was not activated at the DISC. Although total cellular DR4 mRNA and protein were virtually identical in TRAIL-sensitive parental and TRAIL-resistant clones, DR4 protein expression on the cell surface was essentially undetectable in the TRAIL-resistant clones. Moreover, exogenous DR4 and KILLER/DR5 were not properly transported to the cell surface in the TRAIL-resistant cells. Interestingly, TRAIL-resistant cells were resensitized to TRAIL by tunicamycin pretreatment, which increased cell surface expression of DR4 and KILLER/DR5. Our data suggest that tumor cells may become resistant to TRAIL through regulation of the death receptor cell surface transport and that resistance to TRAIL may be overcome by the glycosylation inhibitor/endoplasmic reticulum stress-inducing agent tunicamycin.

Tissue factor activity of SW-480 human colon adenocarcinoma cells is modulated by thrombin and protein kinase C activation.

Expression of tissue factor (TF), a cellular initiator of the extrinsic coagulation cascade, is a feature of many malignant tumours and is intimately involved in the process of metastasis. SW-480 human colon adenocarcinoma cells responded to thrombin (1 U ml(-1)) or phorbol 12-myristate 13-acetate (PMA, 0.1 microM) with a 6.0-fold and a 7.7-fold increase in their procoagulant activity (PCA), respectively, after 4-6 h incubation in serum-free medium. The thrombin-enhanced PCA was significantly inhibited by complexing of thrombin with hirudin, or by serine protease inhibition with 3,4-dichloroisocoumarin. Both effects of thrombin and PMA on PCA in SW-480 cells were blocked by pretreatment of cells with cycloheximide or actinomycin D, indicating that the response required de novo protein and RNA synthesis. The thrombin-enhanced PCA depended on the activation of protein kinase C (PKC) as it was diminished by staurosporine and calphostin C. Moreover, stimulation of SW-480 cells by thrombin or PMA led to a significant increase in TF mRNA within 3 h as measured by the reverse-transcription PCR method, which was also dependent on the activation of PKC. The unaltered decay rate of thrombin-enhanced TF mRNA, evaluated after the addition of staurosporine, suggested that its inhibitory effect occurred at a transcription level. Our data suggest that thrombin enhances TF gene expression and protein synthesis in tumour cells in vitro via PKC activation. The induction of TF expression in tumour cells by thrombin indicates that tumour-associated PCA might have a positive-feedback effect on in vivo local propagation of thrombus by thrombin formation.

AP2 inhibits cancer cell growth and activates p21WAF1/CIP1 expression.

The 52-kD Activator Protein (AP2) is a DNA-binding transcription factor implicated in signalling terminal differentiation. Profound developmental abnormalities have been recently observed in AP2-null mice. The molecular events by which AP2 promotes differentiation or development are, however, unknown. Increased expression of the universal cell cycle inhibitor p21WAF1/CIP1 occurs in growth-arrested terminally differentiating cells. In a search for cellular factors that could activate p21 during phorbol ester (TPA)-induced differentiation, we identified AP2 as a regulator of p21 expression. Mutagenesis of an AP2 DNA-binding site within a p21 promoter-luciferase reporter inhibited its activation by either AP2 transfection or TPA stimulation. Endogenous p21 protein levels were elevated and DNA synthesis was inhibited in AP2 versus control vector-transfected cells. Overexpression of AP2 in HepG2 human hepatoblastoma and SW480 human colon adenocarcinoma cells inhibited cell division and stable colony formation. These results link the differentiation-associated factor AP2 to negative cell cycle and growth control, possibly through p21 activation.

Thrombin enhances the adhesion and migration of human colon adenocarcinoma cells via increased β3 -integrin expression on the tumour cell surface and their inhibition by the snake venom peptide, rhodostomin

The interactions between tumour cells and the microvasculature, including the adhesion of tumour cells to endothelium and extracellular matrix (ECM) as well as their migratory ability, are prerequisites for metastasis to occur. In this study we showed that thrombin is capable of enhancing in vitro tumour cell metastatic potential in terms of adhesive properties and migratory response. Following exposure to subclotting concentrations of thrombin, SW-480 human colon adenocarcinoma cells exhibited increased adhesion to both the endothelium and ECM component (i.e. fibronectin). Likewise, the pretreatment of thrombin enhanced the migratory ability of SW-480 cells. The enhanced adhesion was significantly inhibited by complexing of thrombin with its inhibitor hirudin, or by serine proteinase inhibition with 3,4-DCI, but was unaffected by pretreatment of tumour cells with actinomycin D or cycloheximide. The effect of thrombin resulted in an upregulated cell-surface expression of beta 3 integrins, a group of receptors mediating interactions between tumour cells and endothelial cells, and between tumour cells and ECM. Antibodies against beta 3 integrins effectively blocked both the enhanced adhesion and migration. This thrombin-mediated up-regulation of beta 3 integrins involved the activation of protein kinase C (PKC) as thrombin-enhanced adhesion was diminished by PKC inhibition. Rhodostomin, an Arg-Gly-Asp-containing antiplatelet snake venom peptide that antagonises the binding of ECM toward beta 3 integrins on SW-480 cells, was about 600 and 500 times, more potent that RGDS in inhibiting thrombin-enhanced adhesion and migration respectively. Our data suggest that PKC inhibitors as well as rhodostomin may serve as inhibitory agents in the prevention of thrombin-enhanced metastasis.

Characterization of integrin expression and regulation on SW-480 human colon adenocarcinoma cells and the effect of rhodostomin on basal and upregulated tumor cell adhesion

Integrins are a superfamily of cell surface glycoproteins that mediate cell-extracellular matrix (ECM) and cell-cell adhesion. Immunofluorescence microscopy and flow cytometric analysis using anti-antigrin mAbs as the primary binding ligands demonstrated that the platelet integrin receptor α IIb β 3 , as well as α v β 3,α 5β 1 and α 6β 1 , are present on the surface of SW-480 human colon adenocarcinoma cells. Monoclonal antibodies (mAbs) against α IIb β 3 inhibited unstimulated basal adhesion to fibronectin by approximately 30% and 40%, respectively. The surface immunoreactivity of tumor cells for α IIb β 3 was enhanced by pretreatment (5 min) with a phorbol ester ( 12-O- tetradecanoylphorbol-13- acetate (TPA)) or a lipoxygenase metabolite of arachidonic acid, 12-hdyroxyeicosatetraenoic acid (12-HETE) in a dose- and time-dependent manner. SW-480 cells possess a large intracellular pool of α IIb β 3 , from which the receptor complex translocates to the cell surface following pretreatment with TPA or 12(S)- HETE . This pretreatment enhances adhesion to fibronectin, which is exclusively by α IIb β 3 integrins. Staurosporine was found to block α IIb β 3 up-regulation and enhanced-adhesion. TPA and 12-(S)_- HETE also facilitated the redisitribution of α IIbβ 3 during the enhanced-spreading process. Rhodostomin, an Arg-Gly-Asp-(RGD) containing antiplatelet snake venom peptide, was about 400-times more patent than RGDS at inhibiting control, TPA- or 12-(S)- HETE -enhanced adhesion of SW-480 cells tp fibronectin. The binding of mAbs against α IIb β 3, α v β 3 and α 5 1 was inhibited by pretreatment with rhodostomin, suggesting that rhodostomin binds via its RGD sequence to multiple integrain receptors (i.e., α IIb β 3 , α v β 3 , α 5β 1 ) expressed on the SW-480 cell surface, inhibiting cell adhesion to ECM.

DEVELOPMENT AND CHARACTERIZATION OF AN ADRIAMYCIN-RESISTANT SW480 HUMAN COLON ADENOCARCINOMA CELL-LINE

An adriamycin resistant Sw480 human colon adenocarcinoma cell line (Sw480 Adr) was developed. Characteristics of the Sw480 Adr cells were compared with that of the parent Sw480 (Sw480 WT) cell line. Sw480 Adr cells were cross-resistant to mitoxantrone but not to cisplatin. Glutathione S-transferase enzyme activity as well as glutathione S-transferase pi content were increased 1.5 fold in the resistant cells, whereas P-170 glycoprotein content was increased several hundredfold. Sw480 Adr cells could be partly resensitised towards adriamycin by prior incubation with verapamil (1.65-3.3 mug/ml) and completely resensitised by cyclosporin A (2.5-5.0 mug/ml). Incubation of Sw480 Adr cells with various concentrations of adriamycin (200-1600 nM) during one week, did not change the P-170 glycoprotein levels. In addition, Sw480 Adr cells cultured in adriamycin-free medium for ten weeks (and passaged each week), showed no significant change in P-170 glycoprotein content. These results indicate that the most important mechanism for adriamycin resistance in Sw480 Adr human colon adenocarcinoma cells is overexpression of the P-170 glycoprotein, an ATP-driven drug efflux pump located in the plasma membrane. This overexpression of the P-170 glycoprotein in Sw480 Adr cells does not seem to be reversible.