Colon Carcinoma-derived Cell Line Research Articles

Insights into the limitations of transient expression systems for the functional study of p53 acetylation site and oncogenic mutants

Tumor suppressor protein p53 protects cells against malignant transformation mostly through transcriptional activation. Lysine acetylation is required to mediate activation of p53. The protein displays eight lysine residues and their evolutionary conservation argues for an essential role. The aim of this study was to investigate the significance of individual acetylation sites in mediating p53 functions. Differences in intracellular localization, protein expression levels, and transcriptional activity were investigated by overexpressing acetylation-deficient p53 variants in the colon carcinoma-derived p53 knock-out cell line HCT 116 p53(−/−). We found that not all lysine residues are equally capable of promoting p53’s functions. Individual amino acid mutations or combinations thereof led to altered p53 expression levels, intracellular distribution, or transcriptional transactivation capacity, as compared to the wild-type protein. However, we observed that the choice of protein tag and expression vector could significantly alter obtained results on certain aspects of p53 function.

Loss of LSR affects epithelial barrier integrity and tumor xenograft growth of CaCo-2 cells.

The lipolysis-stimulated lipoprotein receptor (LSR) is a lipoprotein receptor, serves as host receptor for clostridial iota-like toxins and is involved in the formation of tricellular contacts. Of particular interest is the role of LSR in progression of various cancers. Here we aimed to study the tumor growth of LSR-deficient colon carcinoma-derived cell lines HCT116 and CaCo-2 in a mouse xenograft model. Whereas knockout of LSR had no effect on tumor growth of HCT116 cells, we observed that CaCo-2 LSR knockout tumors grew to a smaller size than their wild-type counterparts. Histological analysis revealed increased apoptotic and necrotic cell death in a tumor originating from LSR-deficient CaCo-2 cells. LSR-deficient CaCo-2 cells exhibited increased cell proliferation in vitro and an altered epithelial morphology with impaired targeting of tricellulin to tricellular contacts. In addition, loss of LSR reduced the transepithelial electrical resistance of CaCo-2 cell monolayers and increased permeability for small molecules. Moreover, LSR-deficient CaCo-2 cells formed larger cysts in 3D culture than their wild-type counterparts. Our study provides evidence that LSR affects epithelial morphology and barrier formation in CaCo-2 cells and examines for the first time the effects of LSR deficiency on the tumor growth properties of colon carcinoma-derived cell lines.

The role of CDX2 in Caco-2 cell differentiation

BackgroundCDX2 plays a key part in the differentiation of Caco-2 cells, a colon carcinoma derived cell line that undergoes spontaneous differentiation. The effect of CDX2 expression in Caco-2 cells over time in culture has not been studied yet on a genome-wide level. MethodsThe impact of CDX2 expression on the genomic profile of Caco-2 cells was studied by transducing cells with CDX2 targeting shRNAs. Knockdown efficiency was assessed on mRNA level and protein level by RTPCR, microarrays, and Western blots. Gene set enrichment analysis was performed to assess regulation of specific gene sets. ResultsCDX2 expression had an inhibitory effect on the transcriptional activity of β-catenin/TCF at early stages of culturing, while at later stages, its role in the trans-activation of target genes specific for small intestinal enterocytes seemed more dominant. ConclusionsThe unique induction of a small intestinal signature upon differentiation in Caco-2 cells seems to be at least partially under the control of CDX2.

Bioavailabilty Studies of Curcuminoids: Lessons Learned and the Need for Labeled Reference Materials

Turmeric, the rhizome of the plant Curcuma longa L., has been used for centuries in Ayurvedic medicine for the treatment of different diseases. Curcuminoids – curcumin, demethoxy-curcumin, and bisdemethoxy-curcumin – represent the major active constituents of turmeric. It is essential to determine the bioavailability of curcuminoids, the ability to deliver the active ingredient to the bloodstream. However, since curcuminoids have high hydrolytic instability at physiological pH and poor intestinal absorption, their presence in blood/plasma is very low. Radiolabeled materials are excellent tools to assess the difference between the amount of compound absorbed and metabolized during first pass through the intestine and liver. But, synthesis of radiolabeled analogues is costly and the analysis requires special facility and equipment, therefore the use of labeled compounds in botanical research is very limited. Without available labeled compounds, the bioavailability of curcuminoids is measured using various analytical techniques such as gas chromatography, capillary electrophoresis with amperometric detection, liquid chromatography (LC) with UV or photodiode array detection, LC-tandem mass spectrometry and matrix assisted laser desorption/ionization. Human colon carcinoma-derived cell line Caco-2 can also be used to estimate bioavailability. The apparent permeability coefficient P app is generally acknowledged as predictive for in vivo bioavailability: P app <1 indicates less than 20%, absorption. Our experiments showed that when differentiated Caco-2 cells were exposed to curcumin, the major basolateral products were hexahydro-curcumin sulfate, small amounts of curcumin-glucuronide and octahydro-curcumin sulfate; confirming the rapid reduction of curcumin. For curcumin, a P app of 0.03–0.07 was calculated, indicating virtually no bioavailability of the non-metabolized compound. For metabolites, P app was 0.5–1.3 suggesting low bioavailability of the metabolites.

Hypericum androsaemum water extract inhibits proliferation in human colorectal cancer cells through effects on MAP kinases and PI3K/Akt pathway

MAP kinase and PI3K/Akt signalling pathways are commonly altered in colorectal carcinoma (CRC) leading to tumor growth due to increased cell proliferation and inhibition of apoptosis. Several species of the genus Hypericum are used in Portugal to prepare herbal teas to which digestive tract effects are attributed. In the present study, the antiproliferative and proapoptotic effects of the water extracts of H. androsaemum (HA) and H. perforatum (HP) were investigated in two human colon carcinoma-derived cell lines, HCT15 and CO115, which harbour activating mutations of KRAS and BRAF, respectively. Contrarily to HP, HA significantly inhibited cell proliferation and induced apoptosis in both cell lines. HA decreased BRAF and phospho-ERK expressions in CO115, but not in HCT15. HA also decreased Akt phosphorylation in CO115 and induced p38 and JNK in both cell lines. HA induced cell cycle arrest at S and G2/M phases as well as caspase-dependent apoptosis in both cell lines. Chlorogenic acid (CA), the main phenolic compound present in the HA extract and less represented in the HP water extract, did, however, not show any of those effects when used individually. In conclusion, water extract of HA, but not of HP, controlled CRC proliferation and specifically acted on mutant and not wild-type BRAF. The effect of HA was, however, not due to CA alone.

2-Methoxyestradiol: New perspectives in colon carcinoma treatment

Colon carcinoma represents a major problem in oncology, since this type of cancer responds poorly to conventional chemotherapy. Many groups are actively involved in the search of new experimental strategies to bypass this problem. We investigated the effects of 2-methoxyestradiol (2-ME), which derives from the NADPH-dependent cytochrome P450 metabolism of 17β-estradiol. This compound has raised much interest in the past few decades for its inhibitory effects on the growth of cancer cells of different origin; however, little is known about its use on colon carcinoma-derived cell lines. In the present study, we investigated the effects of 2-ME on cell proliferation and cell cycle of two human colon carcinoma cell lines, namely HCT116 and SW613-B3. Our results showed a net anti-proliferative effect of 2-ME on both cell lines, which is accompanied by cell cycle arrest; moreover, we demonstrated that 2-ME is able to induce apoptosis as well as autophagy. This body of evidence points out that 2-ME could be considered as a promising tool against colon carcinoma.

Gene expression of CYP3A4, ABC-transporters (MDR1 and MRP1-MRP5) and hPXR in three different human colon carcinoma cell lines.

Colon carcinoma cell lines are used widely as screening models for intestinal absorption of drugs. However, the expression of important transport systems and of metabolic enzymes is not completely characterized yet. The expression and inducibility of multidrug resistance gene 1 (MDR1) and cytochrome P450 isoform 3A4 (CYP3A4) was investigated in Caco-2 parental, Caco-2 TC-7 (TC-7) and LS180 cell lines. In the same three cell lines, we investigated the expression of isoforms of the multidrug resistance associated protein family (MRP1-MRP5) and the human pregnane X receptor (hPXR), which may be important for MDR1 and CYP3A4 induction. Cells were treated with rifampicin or 1alpha,25-dihydroxycholecalciferol (1,25(OH)(2)D(3)) for 72 h and the total RNA was extracted. Afterwards reverse transcription real-time polymerase chain reaction (TaqMan) assay was performed to determine the mRNA expression level. We have shown that in LS180 cells, MDR1 and CYP3A4 were inducible with both inducers. In Caco-2 parental and TC-7 cells, CYP3A4 was only inducible with 1,25(OH)(2)D(3). Furthermore, differences were shown in gene expression of several transport proteins (MDR1 and MRP1-MRP5) and CYP3A4 in different human colon carcinoma derived cell lines. hPXR mRNA was expressed in all three cell lines but the amount of mRNA detected was significantly higher in LS180 cells than in Caco-2 and TC-7 cells. We concluded that LS180 cells were a suitable model to study MDR1 and CYP3A4 induction, but for drug transport studies Caco-2 parental and TC-7 cells would be preferred as the more physiological model.

Salvia Fruticosa, Salvia Officinalis, and Rosmarinic Acid Induce Apoptosis and Inhibit Proliferation of Human Colorectal Cell Lines: The Role in MAPK/ERK Pathway

Epidemiological studies have shown that nutrition is a key factor in modulating sporadic colorectal carcinoma (CRC) risk. Aromatic plants of the genus Salvia (sage) have been attributed many medicinal properties, which include anticancer activity. In the present study, the antiproliferative and proapoptotic effects of water extracts of Salvia fruticosa (SF) and Salvia officinalis (SO) and of their main phenolic compound rosmarinic acid (RA) were evaluated in two human colon carcinoma-derived cell lines, HCT15 and CO115, which have different mutations in the MAPK/ERK and PI3K/Akt signalling pathways. These pathways are commonly altered in CRC, leading to increased proliferation and inhibition of apoptosis. Our results show that SF, SO, and RA induce apoptosis in both cell lines, whereas cell proliferation was inhibited by the two sage extracts only in HCT15. SO, SF, and RA inhibited ERK phosphorylation in HCT15 and had no effects on Akt phosphorylation in CO115 cells. The activity of sage extracts seems to be due, at least in part, to the inhibition of MAPK/ERK pathway.

Apoptosis by cisplatin requires p53 mediated p38α MAPK activation through ROS generation

Cisplatin is one of the major chemotherapeutic weapons used against different human cancers, although its mechanism to induce apoptosis is not fully understood. The presence of wild type p53 has been suggested to be important for cisplatin cytotoxicity, hence we found that cisplatin induced apoptosis in cell lines with functional p53. Using the HCT116 colon carcinoma derived cell line we have established that the apoptotic activity of cisplatin requires the onset of a p53-mediated p38alpha MAPK pathway through generation of reactive oxygen species (ROS). HCT116 p53-deficient cells were much less sensitive to apoptosis by cisplatin than their p53wt counterparts, where apoptosis was strongly inhibited by antioxidants. Moreover, the presence of pifithrin-alpha, an inhibitor of p53 transcriptional activity, blocked cisplatin-induced apoptosis, reduced the generation of ROS produced upon cisplatin treatment. In addition, we have identified p38alpha as the isoform necessary for cisplatin-induced apoptosis, upon activation by p53-mediated ROS production. p38alpha MAPK contributes to further activation of p53, which leads to a positive feedback loop, p38alpha MAPK/p53. We conclude that the p53/ROS/p38alpha MAPK cascade is essential for cisplatin-induced cell death in HCT116 cells and the subsequent p38alpha/p53 positive feedback loop strongly enhances the initial p53 activation.

Chemopreventive effects of lupulone, a hop β-acid, on human colon cancer-derived metastatic SW620 cells and in a rat model of colon carcinogenesis

The bitter acids of hops (Humulus lupulus L.) mainly consist of humulones or alpha-acids and lupulones or beta-acids. We aimed to evaluate the antiproliferative mechanisms of lupulones on a human metastatic colon carcinoma-derived cell line (SW620 cells) and to assess their chemopreventive effects in a model of colon carcinogenesis. SW620 cell growth was inhibited by 70% after a 48 h exposure to lupulones (40 microg/ml). Lupulones up-regulated the expression of Fas receptor (Fas) and Fas ligand (FasL) as well as TNF-related apoptosis inducing ligand (TRAIL)-R1 (DR4) and -R2 (DR5) receptor proteins, suggesting the involvement of Fas and TRAIL receptors-mediated pathways in lupulone-induced apoptosis. Lupulones also increased the mitochondrial membrane permeability. Colon carcinogenesis was initiated in Wistar rats by intra-peritoneal injections of azoxymethane (AOM), once a week for 2 weeks. One week after the last injection, rats received lupulones (0.001 or 0.005%) in drinking water, and AOM-control rats received the excipient. After 7 months of treatment, the colon of rats receiving 0.001 and 0.005% lupulones showed, respectively, a 30 and a 50% reduction (P < 0.05) of the number of preneoplastic lesions (aberrant crypt foci). In addition, we observed a drastic reduction (70-80%) of the total number of tumors in the colon of rats treated with lupulones when compared with the AOM control group. Lupulones induced apoptosis in SW620 colon-derived metastatic cells by activating both Fas and TRAIL death receptor signaling pathways, and antagonize at a low dose (4 mg/kg/day) colon cancer development. These observations suggest the use of lupulones for colon cancer chemoprevention trials.

Hypericum perforatum: Which Constituents may Induce Intestinal MDR1 and CYP3A4 mRNA Expression?

In vitro and in vivo studies suggest that extracts of St John's wort (Hypericum perforatum, L .; SJWE) interact with various drugs, by enhancing their elimination, due to induction of intestinal and hepatic cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp), the gene product of multidrug resistance gene 1 (MDR1/ABCB1). The aim of our study was to identify the major constituents responsible for this induction and their relative importance. Therefore, plant extracts were investigated that vary in these constituents with respect to their effect on mRNA expression of MDR1/CYP3A4. First, different pure constituents of Hypericum perforatum L . were investigated. Secondly, diverse SJWE with different concentrations of hyperforin, quercitrin and hypericin were investigated. The concentrations of hyperforin, hypericin, and quercitrin in the plant extracts were determined by HPLC, and an "artificial extract" containing the same mixture of these constituents was investigated. Different plant extracts, pure constituents or "artificial extracts" were applied to the human colon carcinoma-derived cell line (LS180) and the induction of MDR1 and CYP3A4 expression was analyzed by quantitative RT-PCR. MDR1 and CYP3A4 mRNA expression were both induced by single constituents of SJW such as hypericin and hyperforin in a concentration of 10 microM. Additionally, CYP3A4 mRNA expression was induced by quercitrin. SJW extracts containing hyperforin induced significantly MDR1 mRNA expression, whereas no CYP3A4 induction was observed after treatment with any of the investigated SJWE. These effects could be mimicked by "artificial extracts" containing the same compositions of hyperforin, hypericin and quercitrin as the plant extracts.

Phase 2 study of ABT-751 in patients with refractory metastatic colorectal carcinoma (CRC)

3537 Background: ABT-751, an oral antimitotic agent that binds to the colchicine site of β-tubulin and prevents microtubule polymerization, has shown antitumor activity in CRC in xenograft models, including HCT-15, a human colon carcinoma-derived cell line in which paclitaxel and vincristine were ineffective. Minor responses to ABT-751, including one partial response, were seen in CRC patients in phase 1 studies. Methods: This phase 2, multi-center study evaluated the safety and efficacy of ABT-751 in patients with metastatic CRC (N = 49) refractory to irinotecan or oxaliplatin [single refractory (SR) arm, n = 14], or irinotecan and oxaliplatin [double refractory (DR) arm, n = 35]. For each cycle, patients received 200 mg ABT-751 orally once daily for 21 days followed by a 7-day break. Time to disease progression (TTP) was analyzed using Kaplan-Meier Methods: CT scans were obtained every 2 cycles. Patient response was evaluated using RECIST. Results: The median duration of therapy was 2 cycles (range 1–8). 57% of patients experienced Grade 3 or 4 toxicities. 18/49 (37%) subjects experienced Grade 3 or 4 toxicities considered probably or possibly related to study drug, including 15/35 in the DR arm and 3/14 in the SR arm [most common = asthenia (12%), anorexia (10%), neuropathy (8%), and constipation (6%)]. Median TTP was 2.1 months (95% CI = 1.9–2.6). Median TTP by study arm was 2.0 months for the DR arm (95% CI = 1.8–2.3) and 3.8 months for the SR arm (95% CI = 2.0–3.9). Three patients remain on study in the SR arm, including 1 patient in Cycle 6. One patient discontinued after exhibiting stable disease through 8 cycles. No complete or partial responses have been observed to date. Conclusions: ABT-751 was well tolerated in CRC patients. Despite preclinical and phase 1 data suggesting activity, ABT-751 was not active as a single-agent in patients with multiple refractory metastatic CRC. Phase 2 studies in patients with renal, breast, and lung carcinoma are ongoing. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Abbott Laboratories Abbott Laboratories

Paraoxonases (PONs) 1, 2, and 3 are expressed in human and mouse gastrointestinal tract and in Caco-2 cell line: Selective secretion of PON1 and PON2

The paraoxonase (PON) family contains three genes (PON1/2/3) that are believed to be involved in the protection against oxidative stress. PON1 and PON3 are circulating in serum attached to high-density lipoprotein fraction (HDL), whereas PON2 is ubiquitously expressed. The intestine is the second major organ that synthesizes lipoproteins; therefore, we examined PON mRNA expression and protein levels in gastrointestinal biopsies from humans, from C57BL6 mice, and from Caco-2 cells, a colon carcinoma-derived cell line that exhibits properties of intestinal epithelium at differentiation. PON 1/2/3 mRNA and proteins were present in human biopsies with variable expression among different gastrointestinal segments. Only PON2 and PON3 were present in mice. All PON mRNA, proteins, and enzymatic activities were present in Caco-2 cells. Oxidation of CaCo-2 cells with ferrum ascorbate had no significant effect on PON mRNA expression, but it increased paraoxonase and lactonase activity, whereas statinase activity was decreased. We showed polarized secretion of PON1 (basolateral) and PON2 (apical) into Caco-2 culture medium, raising the possibility that intestine is capable of producing and releasing PON1 and PON3 to the circulation, whereas PON2 is released at the brush-border membrane to intestinal lumen where it may perform another yet unclear function.

Ursodeoxycholic Acid Can Suppress Deoxycholic Acid-Induced Apoptosis by Stimulating Akt/PKB-Dependent Survival Signaling

: The nontoxic bile acid ursodeoxycholic acid (UDCA) is reported to be an anti-apoptotic agent with efficacy against a variety of death stimuli including the cytotoxic bile acid deoxycholic acid (DCA). To gain insight into this anti-apoptotic property, we tested UDCA for its ability to protect the colon carcinoma-derived cell line HCT116 against DCA-induced apoptosis. We found that UDCA could suppress DCA-induced apoptosis in a time- and dose-dependent manner and that this effect correlated with Akt phosphorylation. Importantly, UDCA lost its ability to protect cells from DCA-induced cell death when Akt activity was suppressed genetically using a dominant negative Akt mutant or when PI3K activity was inhibited pharmacologically. These results suggest that UDCA can protect HCT116 cells against DCA-induced apoptosis by stimulating Akt-dependent survival signaling.

Interleukin-1beta (IL-1beta) and IL-6 modulate insulin-like growth factor-binding protein (IGFBP) secretion in colon cancer epithelial (Caco-2) cells.

Chronic inflammation is characterised by modifications in cytokine concentrations, whereas growth is mainly dependent on the GH-IGF axis. IGF-I bioavailability is modulated by a family of IGF-binding proteins (IGFBPs). The aim of the present study was to evaluate the interactions among interleukin-1beta (IL-1beta), IL-6 and IGFBP secretion by intestinal cells to assess whether cytokines modulate IGFBP secretion, and in turn IGF-I and IGF-II bioavailability. The human colon carcinoma derived cell line Caco-2 was used as an in vitro model for its capacity to differentiate spontaneously. Experiments were carried out on day 4 (undifferentiated state) and day 14 (differentiated state) after plating. Carcinoembryonic antigen (CEA) was used as a marker of differentiation and increased in the conditioned media (CM) from days 4 to 14 (0.2+/-0.01 ng/ml per 10(5) cells vs 3.3+/-0.2 ng/ml per 10(5) cells, P<0.05). IGFBP-2 and IGFBP-4 secretion decreased concomitantly. Cells were stimulated with IL-1beta and IL-6 at 1, 10 and 50 ng/ml, and with IL-1beta and IL-6 in combination at the same dose of 1 and 10 ng/ml. IGF-I at 50 ng/ml was used as a control. Caco-2 cells expressed and secreted mainly IGFBP-2 and IGFBP-4 into the CM. On day 4, IL-1beta (1 ng/ml) and IL-6 (10 and 50 ng/ml) reduced IGFBP-2 by 29+/-8%, and by 32+/-9 and 38+/-8% respectively (P<0.05). IGFBP-4 was also reduced by IL-1beta at 1 and 50 ng/ml (-14+/-4% and -46+/-11% vs serum free medium (SFM) respectively, P<0.05), and IL-6 at 50 ng/ml (-46+/-15%, P<0.05). Both IGFBP-2 and IGFBP-4 were reduced by IL-1beta and IL-6 in combination at 1 and 10 ng/ml (P<0.05). On day 14, IGFBP-2 band intensity was reduced at 10 ng/ml of IL-1beta (-22+/-15% vs SFM, P<0.05) and at 50 ng/ml of both cytokines (-33%+/-8% and -13%+/-13% vs baseline respectively, P<0.05). IGFBP-4 band intensity decreased with 10 and 50 ng/ml of IL-1beta (-35+/-11% and -46+/-15% vs SFM respectively) and IL-6 (-36%+/-10% and -46+/-15% vs SFM respectively). IL-1beta and IL-6 in combination at 1 and 10 ng/ml reduced both IGFBP-2 and IGFBP-4.In conclusion, IGFBP-2 and IGFBP-4 secretion in CM decreased with Caco-2 cell differentiation. IGFBP-2 and IGFBP-4 were significantly decreased by IL-1beta and IL-6 treatment in both the undifferentiated and differentiated state. Furthermore, these cytokines increased cell proliferation whereas total protein content was significantly reduced only at the higher concentrations of IL-6 and IL-1beta. These findings suggest that interleukins modulate the IGF-IGFBP system in Caco-2 cells in vitro.

Polyamine metabolism in primary human colon adenocarcinoma cells (SW480) and their lymph node metastatic derivatives (SW620).

The natural polyamines are multifunctional constituents of all eucaryotic cells. The objective of this work was to compare aspects of polyamine metabolism in two related cell lines with the idea to investigate whether metabolic differences can be attributed to functional differences of the cells. The human colon carcinoma-derived cell lines SW480 and SW620 were chosen as models. SW480 cells were isolated from the primary tumour, SW620 cells from a lymph node of the same patient. SW620 cells grow faster, and the key regulatory enzymes of polyamine biosynthesis (ODC and AdoMetDC) are more active in the metastatic cells. Moreover, their ability to accumulate polyamines from the environment is more important than of SW480 cells. Likewise polyamine concentrations were markedly higher in SW620 cells, although they are much smaller than SW480 cells, and have a particularly small cytoplasmic space. Both cell lines show a striking diminution of ODC and AdoMetDC activities and changes in the polyamine patterns at the transition from exponential to non-exponential growth--most probably as a consequence of high cell density. Depletion of putrescine and spermidine due to inactivation of ODC by DFMO causes accumulation of cells in G1, and a proportional decrease of S-phase cells in both cell lines. Based on morphologic and other criteria SW480 and SW620 cells were typified as poorly differentiated. In agreement with their low grade of differentiation they exhibit a low alkaline phosphatase activity. However, the time-dependent decrease of alkaline phosphatase is not typical of differentiation patterns of other adenocarcinoma-derived cell lines or of normal enterocytes. The high capacity of de novo polyamine biosynthesis and of polyamine uptake is presumably a prerequisite for the rapid growth and invasiveness. The fact that these properties were more accentuated in the case of SW620 cells and paralleled enhanced metastatic properties indicate relationships between basic parameters of polyamine metabolism and malignancy.

Anti-metastatic effect of the sialyl Lewis-X analog GSC-150 on the human colon carcinoma derived cell line KM12-HX in the mouse.

We investigated the inhibitory effect of the sialyl Lewis-X (sLeX) analog, GSC-150, on hepatic metastasis of the human colon carcinoma derived cell line, KM12-HX, which highly expresses sLeX antigen on the cell surface. The number of cancer nodules found in BALB/c nude mouse liver 6 weeks after intrasplenic injection of KM12-HX cells was significantly reduced by co-administration of GSC-150. The amount of [3H]thymidine-labeled KM12-HX cells distributed in liver was also significantly reduced by GSC-150 co-administration in lipopolysaccharide (LPS)-treated mice at 48 h after administration of the tumor cells, while GSC-150 did not reduce the amount of HX cells distributed at 30 min. Considering our previous report that the initial phase of the distribution of KM12-HX cells in liver is governed by their being trapped in the hepatic microvessels because of their large size (Mizuno et al., J. Hepatol., 28, 865-877, 1998), these results suggest that GSC-150 does not inhibit this first-pass trapping by microvessels, but inhibits the subsequent process which is more directly related to final metastasis. GSC-150 inhibited the adhesion of KM12-HX cells to tumor necrosis factor-alpha (TNF-alpha)-activated human umbilical vein endothelial cells (HUVECs). These findings imply that the anti-metastatic effect of GSC-150 in vivo could be explained by its inhibition of cell-cell interactions between cancer and host cells.

Effect of the polyamine oxidase inactivator MDL 72527 on N1-(n-octanesulfonyl)spermine toxicity

N 1-(n-octanesulfonyl)spermine ( N 1OSSpm) is a potent calmodulin antagonist. In the present work, its toxicity to DHD/K12/TRb and CaCo-2 cells, two colon carcinoma-derived cell lines, was studied with the aim to identify those properties of the cells, which determine their sensitivity to N 1OSSpm and related structures. Exposure of the cells to MDL 72527, a compound considered to be a selective inactivator of polyamine oxidase (PAO) increased the cytotoxicity of N 1OSSpm to both cell lines. In contrast, toxicity of trifluoperazine, a calmodulin antagonist with a polyamine-unrelated structure, was not enhanced by MDL 72527. Combined exposure of cells to 2-(difluoromethyl)ornithine (DFMO) (a selective inactivator of ornithine decarboxylase), MDL 72527 and N 1OSSpm produced a synergistic cytotoxic effect. Neither the intrinsic PAO activity of the cells (as determined with N 1, N 12-diacetylspermine as substrate), nor their ability to accumulate the drug was a determinant of the cytotoxic effect of N 1OSSpm. These data suggest that MDL 72527 has a target unrelated to PAO, which is responsible for the enhancement of N 1OSSpm (and spermine) toxicity. Identification of this target may be of use if the therapeutic potentials of MDL 72527 are to be exploited.

Inhibition of polyamine oxidase enhances the cytotoxicity of polyamine oxidase substrates. A model study with N 1-( n-octanesulfonyl)spermine and human colon cancer cells

N 1-( n-octanesulfonyl)spermine (N 1 OSSpm) is a substrate of polyamine oxidase. It shares several properties with spermine, such as antagonism of NMDA-type glutamate receptors, calmodulin antagonism, and cytotoxicity, but it is more potent by orders of magnitude in these regards than spermine. The human colon carcinoma-derived cell line CaCo-2 was used as a model to study the toxicity of N 1 OSSpm as a function of polyamine oxidase (PAO) activity and differentiation. If the formation of hydrogen peroxide and aminoaldehyde by the PAO-catalysed reactions was prevented by selective inactivation of the enzyme with MDL 72527, cytotoxicity of N 1OSSpm was not diminished, but on the contrary, enhanced. Exponentially growing CaCo-2 cells were considerably more sensitive to N 1OSSpm than differentiating cells. The results suggest that cytotoxic substrates of PAO exhibit enhanced cytotoxicity in cells, if PAO activity is inhibited. Since tumour cells are known to have lower polyamine oxidase activities than their normal counterparts, it will be interesting to explore whether cytotoxic substrates of polyamine oxidase, for which N 1OSSpm is an example, are suited to preferentially kill tumour cells.

In vitro effects of melatonin on cell proliferation in a colon adenocarcinoma line.

The effect of melatonin on inhibition of cell growth was studied in CT-26, a murine colon carcinoma-derived cell line. Cells growing in exponential phase were exposed to low (10(-7)-10(-10) M) and high doses (1, 2 and 3 x 10(-3) M) of melatonin during 24 h. Synthesis of DNA was measured by 5-bromo-2'-deoxyuridine incorporation. There was no effect at low doses, but a statistically significant correlation was found between the decrease in DNA synthesis and the dose of melatonin used (r = -0.52, P < 0.001). This implied the following percentages of inhibition: 1 mM, 22%; 2 mM, 25%; 3 mM, 47%. Potential cell membrane damage by high doses of melatonin was investigated by lactate dehydrogenase measurement and no significant levels were observed. Analysis with a single saturation technique showed no detectable oestradiol receptors in this cell type; therefore, we can assume that the effects occurring with the addition of melatonin were not mediated by modulation of this hormone on oestrogen receptors. The decreases in cell growth were attributed to a moderate, but significant antiproliferative action of melatonin on this non-hormone-dependent cell line.