Selective toxicity of 1-naphthol to human colorectal tumour tissue

1-Naphthol was selectively toxic to human colorectal tumours compared to corresponding normal colonic tissue removed at surgery and maintained in short-term organ culture. Nineteen of 24 tumours studied have shown a significant differential response. Three human colonic adenocarcinoma xenografts, in the short-term organ culture system, displayed the same response to 1-naphthol as primary tumours removed at surgery. 1-Naphthol, 1,2- and 1,4-naphthoquinone were also toxic to two human colonic adenocarcinoma cell lines, LoVo and COLO 206. The selective toxicity of 1-naphthol is mediated in part through an accumulation of 1-naphthol in the tumour tissue due to impaired conjugation by the tumour. The higher concentrations of 1-naphthol may then exert their toxicity either directly or by formation of naphthoquinones. Some indirect evidence was obtained for the possible involvement of 1,2- or 1,4-naphthoquinone in the cytotoxicity of 1-naphthol. Our studies suggest that further studies are warranted of the possible use of 1-naphthol or related compounds as antitumour agents.     

Metabolism of benzo(a)pyrene and 1-naphthol in cultured human tumorous and nontumorous colon

The oxidative metabolism of benzo(a)pyrene and the conjugative metabolism of 1-naphthol by explant cultures of normal human colon and colonic tumor tissue, obtained at surgery, have been studied. After 24 hr in culture, the explants were exposed to either [1-14C]-1-naphthol (20 to 100 microM) or [3H]-benzo(a) pyrene (1.5 microM) for a further 1.5 to 24 hr. Both normal-appearing tissue and tumor tissue metabolized benzo(a)pyrene to a wide variety of organic solvent-soluble metabolites, including monohydroxybenzo(a)pyrenes, dihydrodiols, and tetrols. 1-Naphthol was metabolized by cultured human colonic mucosa and tumor tissue to both its glucuronic acid and sulfate ester conjugates. In the normal tissues, with naphthol (20 microM), sulfate ester conjugation predominated. However, with the tumor tissue, sulfate ester conjugation decreased; thus, the percentage of glucuronic acid conjugates, expressed as a percentage of total metabolites formed, was increased significantly compared to normal tissue. The relationship, if any, of these changes to neoplastic transformation is unclear. The technique of explant culture described in this study may be of use for the study of other facets of the pathobiology of solid tumors.     

Differential glycosylation of MUC1 and CEACAM5 between normal mucosa and tumour tissue of colon cancer patients

Altered glycosylation in epithelial cancers may play an important role in tumour progression, as it may affect tumour cell migration and antigen presentation by antigen presenting cells. We specifically characterise the glycosylation patterns of two tumour antigens that are highly expressed in cancer tissue and often detected in their secreted form in serum: the epithelial mucin MUC1 and carcinoembryonic antigen (CEA, also called CEACAM5). We analysed 48 colorectal cancer patients, comparing normal colon and tumour epithelium within each patient. Lectin binding was studied by a standardised CEA/MUC1 capture ELISA, using several plant lectins, and the human C-type lectins MGL and DC-SIGN, and Galectin-3. Peanut agglutinin (PNA) bound to MUC1 from tumour tissue in particular, suggests increased expression of the Thomsen-Friedenreich antigen (TF-antigen) (Core 1, Galβ1-3GalNAc-Ser/Thr). Only small amounts of Tn-antigen (GalNAcα-Ser/Thr) expression was observed, but the human C-type lectin MGL showed increased binding to tumour-associated MUC1. Furthermore, sialylation was greatly enhanced. In sharp contrast, tumour-associated CEA (CEACAM5) contained high levels of the blood-group related carbohydrates, Lewis X and Lewis Y. This correlated strongly with the interaction of the human C-type lectin DC-SIGN to tumour-associated CEA, suggesting that CEA can be recognized and taken up by antigen presenting cells. In addition, increased mannose expression was observed and branched N-glycans were prominent, and this correlated well with human Galectin-3 binding. These data demonstrate that individual tumour antigens contain distinct glycan structures associated with cancer and, since glycans affect cellular interactions with its microenvironment, this may have consequences for progression of the disease.     

Determinants of O(6)-alkylguanine-DNA alkyltransferase activity in normal and tumour tissue from human colon and rectum

O(6)-Alkylguanine-DNA-alkyltransferase (ATase) is an important modulator of alkylating agent-induced toxicity and carcinogenicity, but those factors which influence the expression of this repair protein in human tissues are poorly characterised. In this study, we have determined ATase levels in macroscopically normal and tumour tissues from 76 individuals with benign or malignant colorectal disease. All tissue samples had detectable ATase activity, with values ranging from 35 to 451 fmol/mg protein. ATase activity in normal rectal tissue was significantly higher than that in normal tissue from the sigmoid colon (148 +/- 76 vs. 100 +/- 40 fmol/mg protein, p = 0.01), whereas ATase levels within different regions of the colon (proximal vs. sigmoid colon) were similar. In normal tissue, inter-individual variation in ATase activity was 4-fold in the colon and 6-fold in the rectum, whereas in tumour tissue the corresponding figures were approx. 13.0- and 7-fold, respectively. There was no detectable difference in normal tissue ATase activity between individuals with benign or malignant disease of the colon. Normal and tumour tissue ATase activities were strongly correlated in the sigmoid colon (r = 0.80) and rectum (r = 0.59) but not the caecum (r = -0.03). In a multivariate analysis, ATase activity in normal colon tissue increased with age (p = 0.01) and current smoking (p = 0.06), whereas tumour ATase activity increased only with use of anti-histamines (p = 0.05). In rectal tumour tissue, activity decreased with age (p = 0.05) and use of anti-muscarinic medications (p = 0.01): in normal rectal tissue, no modulating factors were identified.     

Assessment of human tumour proliferation using bromodeoxyuridine--current status.

The study of human tumour proliferation has been facilitated by the development of monoclonal antibodies recognizing halogenated pyrimidines. Flow cytometry can be used to detect the incorporation of bromodeoxyuridine (BUdR) into DNA simultaneously with the measurement of total DNA content. We have studied in excess of 600 tumours using in vivo administration of BUdR. The cell kinetic information generated from this approach is more complete than can be obtained from in vitro incubation with DNA precursors such as tritiated thymidine (3HTdR) or by single parameter DNA analysis. The duration of S-phase (Ts) can be estimated in addition to the labelling index (LI). From these two parameters, the potential doubling time (Tpot) can be calculated. Our data show a wide variation in Ts as well as LI, making both these parameters important variants in determining overall proliferation. Although there is tremendous variation in Tpot between tumours of the same and different types, the median values are surprisingly short at around 5 days. A close relationship exists between the presence of DNA aneuploidy and the proliferation parameters. The clinical relevance of Tpot is currently being assessed independently in two trials of accelerated versus conventional fractionation.     

Characterization of human colon carcinoma cell lines isolated from a single primary tumour.

The initiation of a cultured human colon carcinoma line on a feeder layer of confluent fibroblasts is described. Attempts to initiate cultures without fibroblast feeder layers were not successful. Two sub-lines (designated HCT C and HCT C Col) were isolated and weaned from cells growing on the surface of the feeder layer. The sub-lines had different morphologies, secreted different levels of carcinoembryonic antigen (CEA) into the medium of confluent cultures and had slightly different karyotypes. Both sub-lines grew in semi-solid medium and formed xenografts when injected s.c. into athymic nude mice. Analysis of radioiodinated cell membrane components indicated small, but significant differences between the sub-lines.     

Glutathione S-transferases in normal and cancerous human colon tissue

In order to evaluate the role of the placental form of glutathione S-transferase (GST-Pi) as a tumour marker, activity andcomposition of GSTs from human colon were investi gated. GSTswere purified from normal colon mucosa and from colomc tumoursby affinity chromatography on gluta thione-agarose. After SDS-PAGEor isoelectric focusing these purified preparations revealedonly one band that comigrated with GST-Pi from human placenta.A monoclonal antibody (mAb) very specific for GST-Pi was developedand characterized. On inimunoblot this mAb stains purified GSTfrom normal and diseased colon tissue. GST activity was significantlyhigher in most cancerous (247 ? 38 nmoll mm/mg protein; n =7), compared with the corresponding normal tissues (171 ? 18nmol/min/mg protein; n = 7). In colon from patients withoutlarge bowel malignancies GST Pi is also by far the most prominentisoform detectable. In conclusion, both normal and tumorouscolon tissue predominantly express GST-Pi and therefore GST-Piis not suitable as a tumour marker for colonic carcinomas. However,the increased GST-Pi levels in colonic tumours could possiblycontribute to the relatively high resistance to anti-cancerdrugs.     

Extensive liver metastasis from human colon cancer in nude and scid mice after orthotopic onplantation of histologically-intact human colon carcinoma tissue.

Clinically-relevant animal models of human cancer are greatly needed for the study of human cancer biology and the development of new cancer therapeutics and diagnostics. We report here that by orthotopically transplanting histologically-intact human colon cancer to the colon of the immunodeficient nude and scid mouse mutants that extensive local growth and liver metastases occur consistently even after extensive in vivo orthotopic passage. We demonstrate that the liver metastases arise by hematogenous spread. The models described in this report for human colon cancer should prove useful for individual cancer patients as well as for basic and applied studies to develop improved treatment.     

Inhibition of lymphocyte cytotoxicity for human colon carcinoma by treatment with solubilized tumour membrane fractions

The in vitro cytotoxic action of patients' lymphocytes against colon carcinoma cells was evaluated following incubation of the lymphocytes with papain-solubilized tumour-membrane preparations. Soluble extracts of pooled colon carcinomas inhibited cytotoxicity by sensitized lymphocytes, but similar extracts of normal colon or melanoma had no inhibitory effect. The results suggest that soluble tumour antigen may play a role in abolishing lymphocyte reactivity, and this is interpreted as supporting the concept that cellular immunity against tumours in vivo may be inhibited by circulating antigen.     

Effects of lonidamine alone or combined with hyperthermia in some experimental cell and tumour systems.

Lonidamine or 1-[(2, 4-dichlorophenyl) methyl]-1H-indazole-3-carboxylic acid, studied in a battery of in vitro and in vivo tests currently used for the screening of anti-tumour agents affecting cell division, has been shown to have a narrow spectrum of anti-tumour activity. The significance of this finding is discussed in the light of previous investigations suggesting that lonidamine affects mitochondrial function and not cell replication. Hyperthermia has been shown to sensitize tumour cells to lonidamine. This observation indicates that in combination with hyperthermia lonidamine has some potential for the treatment of cancer; moreover, it suggests that hyperthermia might reproduce a metabolic condition occurring in some stages of the disease. The blood levels corresponding to the anti-tumour action of lonidamine in animals are in the range of those detected in patients treated with the drug.     

Extraction of CEA from tumour tissue, foetal colon and patients' sera, and the effect of perchloric acid.

The use of perchloric acid and water for the extraction of CEA from tumour and foetal tissues has been investigated. In the case of tumour, lower recoveries of CEA were obtained from perchloric acid extracts than from aqueous extracts of the same tissue. CEA has also been extracted with 3M KCl solution from insoluble perchloric acid residues of tumour homogenates and cancer patients' serum. Whilst a large proportion of CEA activity recovered from tumour was associated with the perchloric acid residue, the corresponding amounts from serum were very small. CEA elution volumes for each extract, obtained by assay of Sephadex G-200 column fractions, showed significant heterogeneity in molecular size. The purified CEA pools also showed quantitative variations in the binding profiles on Con A-Sepharose. It has been shown that perchloric acid modifies the carbohydrate in CEA, thus altering its Con A-binding properties. Preliminary experiments with foetal colon have demonstrated that, unlike colorectal CEA, a significant proportion of foetal CEA was not bound to ConA. Comparative immunodiffusion showed immunological identity of CEA from the various extracts, although the purified aqueous extract produced an additional precipitin reaction, indicating a second antigen which is relatively unstable or less soluble in perchloric acid.     

Intra-luminal tumour cells and peri-anastomotic tumour growth in experimental colonic surgery.

Most loco-regional tumour recurrences following colorectal cancer surgery are extra-luminal. This study was designed to determine the conditions necessary for viable intra-luminal tumour cells to give rise to extra-luminal tumour growth in an experimental animal model. When carcinoma cells were injected into the colonic lumen in the absence of a colonic wound, tumour growth was rare (1 of 24 animals). However, when tumour cells were injected intra-luminally either in the presence of an apparently watertight colotomy repair or prior to the transmural insertion of interrupted sutures, peri-anastomotic +/- widespread peritoneal tumour growth was consistently observed. Tumour growth did not occur on the mucosa of the bowel. These findings suggest that viable intra-luminal tumour cells can migrate across a clinically intact experimental large bowel anastomosis or along transmural suture tracts to give rise to extra-luminal tumour growth.     

Effect of diverse categories of drugs on human colon tumour cell proliferation

The effect of a number of drugs belonging to different therapeutic categories on cell proliferation in a human colon cancer cell line (HT 29) was studied. Generally, drugs classified as calcium antagonists had a moderate to strong inhibitory effect on cell growth. The EC50 value for prenylamine, perhexiline and bepridil was 10 microM. The so-called calcium channel blockers (verapamil, nifedipine, diltiazem) were less effective. With the exception of the sodium selective ionophore, monensin, which was extremely potent (EC50 less than 0.1 microM), calmodulin antagonism appeared to be a common feature of compounds inhibiting cell proliferation of human colon tumour cells.     

Expression of selenium-containing proteins in human colon carcinoma tissue

Selenium may be beneficial in reducing the risk of cancer incidence and mortality in many cancer types such as liver, prostate, colorectal and lung. However, despite the extensive recent research on selenium and selenium-containing proteins, there are still open questions concerning their expression in certain human cancer types, including colorectal carcinoma. Therefore, the expression level of the selenoproteins thioredoxin reductases 1 and 2 (TRXR-1 and TRXR-2) and glutathione peroxidases 1 and 4 (GPX1 and GPX4) in human colon carcinoma tissues was investigated. Up-regulation of TRXR-1 in the colon carcinoma specimens was found both in disease stage-dependent and independent analyses. No differences were found for TRXR-2 expression levels. GPX1 was up-regulated in carcinoma tissues at both the protein and mRNA levels. GPX4 was also up-regulated at the protein level, except for the samples derived from stage III patients. The expression of TRXR-1, GPX1 and GPX4, but not TRXR-2 is differently regulated in cancer as compared to healthy colonic tissue.