Enhancement of gastric mucus phospholipid secretion by an antiulcer agent, ebrotidine.

1. Rat gastric mucosal cells, subjected to phospholipid labeling by incubating the cell suspension in DMEM with [3H]choline, were exposed to different concentrations (0-150 microM) of H2-receptor antagonists, ebrotidine and ranitidine, and the phospholipid secretory responses were evaluated. 2. In the absence of the drugs, the secretion of choline-containing phospholipids over a 1 hr period averaged 3.97% of the total cellular labeled phospholipids. Ebrotidine caused a dose-dependent increase in the rate of phospholipid secretion which was most pronounced at 1 hr and persisted for at least 2 hr. The maximal effect was attained at 120 microM ebrotidine giving a 36% increase in phospholipid secretion. 3. The phospholipid secretory response to ebrotidine was accompanied by an increase in gastric mucosal cell cAMP level which reached a maximum value of 2.1-fold over that of controls at 1 hr. Ranitidine, in contrast, neither evoked increase in cAMP level nor caused any stimulation in phospholipid secretion. 4. The results indicate that the gastroprotective properties of ebrotidine are associated with the ability of the drug to elicit a rapid stimulation in gastric mucus phospholipid secretion, and that ranitidine does not possess such property.

Metastasis of human colon tumor cells in vivo: correlation with the overexpression of plasminogen activators and 72 kDa gelatinase.

Metastatic spread of tumor cells depends upon intravasation of malignant cells from the primary site and extravasation into the distant organs following remodeling of the basement membrane. We have investigated the metastatic potential of five tumorigenic human colon carcinoma cell lines, LS 174T, SW 620, WiDr, SW 480 and Caco-2 using intrasplenic injection in nude mice. LS 174T is most aggressive causing liver metastasis in all animals within 6 weeks. SW 620 and WiDr produced liver metastasis in 70% and 30% of the animals but after a period of 12 weeks whereas SW 480 and Caco-2 were not metastatic. LS 174T exhibited high cell-associated urokinase-type plasminogen activator (u-PA) and high secreted u-PA and tissue plasminogen activator (t-PA) levels. WiDr, SW 480 and Caco-2 had essentially similar low levels of cell associated u-PA but WiDr had higher secreted u-PA levels as comprated to the SW 480 and Caco-2 cells. The level of secreted MMP-2 (72 kDa gelatinase) was highest in the most metastatic cell line, LS 174T, and lower in other less metastatic ones. These data show that metastatic behavior of human colon tumor cells correlates with the enhanced secretion of plasminogen activators and MMP-2 by these cells.

Effects of exogenous transglutaminase on spreading of human colorectal carcinoma cells.

Using pre-confluent cultures of a human colon tumor cell line deficient in transglutaminase (LS174T cells), we have investigated the effect of adding exogenous transglutaminase (TGA) on cell spreading. The cells were plated at either 4.5 x 10(5) cells per well (low-seeded cultures) or 9 x 10(5) cells per well (high-seeded cultures) in 24-well dishes and treated for either 1 or 4 days (low- and high-seeded cultures respectively) under following conditions: Chee's Essential Medium (CEM) + 10% fetal calf serum (FCS); CEM + 10% FCS + TGA; CEM + 10% FCS + dithiothreitol (DTT) + CaCl2; CEM + 10% FCS + DTT + CaCl2 + TGA. Photomicrography of the cells after these treatments revealed that in both low- and high-seeded cultures, TGA inhibited the spreading of the cells both in the presence and absence of DTT and calcium. Individual colony sizes were significantly smaller in the presence of TGA. This phenomenon may be related to the ability of TGA to promote cell interactions with the underlying tissue matrices and metastasis.

Sensitivity of human colon tumor metastases to anticancer drugs in athymic (nude) mice.

We have studied the metastatic behavior of five human colon tumor cell lines (LS174T, WiDr, Caco-2, SW 620 and SW 480) using intrasplenic-nude mouse (ISMS) and intravenous-nude mouse (IVMS) model systems. LS174T was highly metastatic in both systems. In the IVMS system, LS174T cells produced lung metastasis and also grew in the skin as if the cells had been injected subcutaneously. We have also studied the anti-metastatic activity of three anticancer drugs (5-fluorouracil (5-FU), doxorubicin (DX) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) using LS174T cells and both ISMS and IVMS systems. The drugs were given intravenously on day 19 and 26 of tumor cell injection. Mice were sacrificed and organs observed for metastatic growth 4-6 weeks after cell injection. The results show that in the ISMS system, BCNU and 5-FU are inactive against both the liver metastasis and primary growth in the spleen. DX inhibits metastatic growth but not the primary growth. In the IVMS system, BCNU is inactive, whereas 5-FU and DX are active against the metastatic growth. Thus, DX may have activity against blood-borne human colon tumor metastasis.

Pharmacological alterations of cellular transglutaminase activity and invasiveness in human colorectal carcinoma cells.

Human colorectal tumor cells expressing differing metastatic potential and tissue transglutaminase (TGA) activity were tested for the ability of various pharmacological agents to enhance TGA activity. The most effective stimulant was tetradecanoylphorbol-13-acetate (TPA), which in human colon carcinoma cells (SW620) caused a 5-fold, protein synthesis dependent increase in activity over 3 days. In WiDr and SW480 cells TGA activity was less susceptible to induction by TPA, possibly owing to the higher basal levels of TGA. Retinoic acid and a synthetic retinoid, [RO 15-1570; (E)-4-[2(5,6,7,8-tetramethylnaphthalene-2-yl)propen-1-yl] benzenesulphonyl-ethane)], also induced TGA activity to a lesser extent in SW620 cells, whereas other differentiation inducers [sodium butyrate and hexamethylene bis-acetamide (HMBA)] were ineffective. In LS174T cells, TGA activity was resistant to induction by all of the agents. The synthetic retinoid (RO 15-1570) inhibited in vitro invasiveness of SW620 cells, however, TPA treatment or addition of exogenous TGA did not inhibit invasiveness of these cells. Hence, the invasive behavior of a metastatic human colon tumor cell line (SW620) does not appear to be dependent on the TGA activity which the cells express. The anti-invasive activity of the retinoid in SW620 cells therefore may be mediated by some other mechanism.

Transglutaminase activity in human colorectal carcinomas of differing metastatic potential.

Transglutaminase (TGA) activity in four human colorectal carcinoma cell lines of differing metastatic potential, and the effects of mild proteolysis on this activity, was investigated. Rank order of metastatic activity measured in nude mice (intrasplenic injection) was found to be LS174T greater than SW620 greater than WiDr greater than SW480. Rank orders of TGA activity were SW480 greater than WiDr greater than SW620 greater than LS174T. Proteolysis of cell lysates increased LS174T TGA activity 42-fold, SW620 2-fold without affecting WiDr or SW480 activity. Hence a negative association exists between metastatic potential and TGA activity in human colorectal carcinoma cells. Furthermore, a positive association exists between proteolytic activation of TGA and metastatic potential.

In vitro response of a human colon tumor xenograft and a lung adenocarcinoma cell line to alpha-difluoromethylornithine alone and in combination with 5-fluorouracil and doxorubicin.

We have investigated effects of alpha-difluoromethylornithine (DFMO), both as a single agent and in combination with 5-fluorouracil (5-FU) against a human colon tumor xenograft (T6) grown as primary tissue culture in serum-free medium and in combination with doxorubicin (DX) against a human lung adenocarcinoma cell line (A549). DFMO showed a dose-dependent growth-inhibitory effect against human colon tumor xenograft and lung adenocarcinoma cells. Growth-inhibitory activity of DFMO against T6 cells was reversed completely when the cells were treated simultaneously with putrescine (PU) (10(-6) M) and DFMO (10(-3) M). When 5-FU and DFMO were used in combination against T6 cells, no antagonism or synergism between the two drugs was seen. However, in the case of A549 cells, when DFMO was used in combination with DX, there was a consistent increase in growth inhibition that surpassed the inhibition of either agent given alone.

Proteins secreted by Caco-2 cells support growth of other tumor cells in Chee's Essential Medium without supplements.

The growth promoting effect of several hormones and growth factors on two human colon tumor cell lines (Caco-2 and SW 48) was studied using six different chemically defined serum-free media (SFM). Caco-2 grew in a simple SFM [GF3: Chee's Essential Medium (CEM) plus insulin, transferrin and selenium], whereas, SW 48 cells did not grow in GF3 medium. This suggested that Caco-2 cells probably secrete proteins in SFM which influence attachment and growth of Caco-2 and other tumor cells. Lyophilized Caco-2 conditioned medium and substratum, when added to plain CEM, supported growth of SW 48 and SW 948 cells. The substratum material was more effective than conditioned medium in promoting growth of the cell lines. The substratum material helps attachment and spreading of the cells and, thus, improves growth of the cells over conditioned medium. Caco-2 conditioned medium and substratum were analyzed for their components using SDS-PAGE system and gel filtration chromatography. The substratum was analyzed for the presence of fibronectin and laminin by the ELISA technique. The conditioned medium does not contain TGF alpha and TGF beta. The growth stimulating activity of the conditioned medium is due to a protein component, approximately 58Kd in size.

Precancerous lesions and biologic markers in esophageal cancer.

Neoplastic development in the esophagus is characterized by abnormal activity in the basal cell proliferative compartment. Malignancy starts with mild dysplasia. Barrett's esophagus is associated with columnar epithelial dysplasia and presents increased risk for adenocarcinoma. Esophagitis, especially with chronicity, may constitute a predisposing characteristic in high incidence areas. This paper briefly describes the main features of precancerous lesions in the esophagus. It then focuses on a discussion of the biological markers of malignancy in the oral cavity and esophagus that are currently under study. Such biomarkers include promising differentiation markers such as keratins, involucrin, particulate transglutaminase, as well as growth factors, and most studied but nonspecific onco-developmental markers, e.g., carcinoembryonic antigen, alpha 1-fetaprotein, hormone/enzyme markers, e.g., human chorionic gonadotropin and placental lactogen, and a number of other miscellaneous markers.

Development of serum-free media for the growth of human gastrointestinal adenocarcinoma xenografts as primary tissue cultures.

The growth-promoting effect of several hormones and growth factors on eight human colon tumor cell lines (SW 48, SW 403, SW 480, SW 620, SW 948, HT29, LS174T and Caco-2) was studied using seven different chemically defined serum-free media [GF3: Chee's essential medium plus insulin, transferrin and selenium; GF3F: GF3 plus fetuin; GF4: GF3 plus linoleic acid/bovine-serum albumin (BSA); GF5: GF4 plus fetuin, GF5E, GF5 plus EGF; GF5T: GF5 plus triiodothyronine; GF7: GF3 plus EGF, transferrin, insulin, linoleic acid/BSA, oleic acid/BSA and fetuin]. GF5 appears to be the best serum-free medium as it supported continuous growth of all of the colon tumor cell lines. GF5 also supported growth of five of the seven human colon and stomach tumor xenografts as primary tissue cultures. However, the stomach xenograft cells had a very slow growth rate as compared to the colon xenograft cells in the medium. Cells grown in GF5 retained their tumorigenicity in athymic (nude) mice and characteristic cellular morphology. GF7 was the poorest of all of the serum-free media studied as none of the cell lines or xenografts grew in this medium.

Tumorigenicity of EJ-ras oncogene transformed NIH 3T3 cells and expression of plasminogen activators.

A number of clonal cell lines have been isolated from NIH 3T3 cells transfected with the plasmid, pSV2 gpt-EJ-ras. The plasmid expresses Val12 instead of Gly12 in p21 ras protein and can be selected for the expression of E. coli XGPRT gene in mammalian cells. Southern analyses of the Eco R1 and Bam H1 digests of chromosomal DNA shows that multiple copies of the plasmid are integrated in a tandem sequence in the clones used in this study. The transfectants showed refractile appearance and criss-crossed pattern of growth, exhibited elevated expression of ras mRNA and formed tumors in nude mice commensurate with the copy number of the integrated EJ-ras gene. The increased propensity to form tumors did not correlate with the expression of urinary or tissue plasminogen activators (u-PA or t-PA). The cellular and secreted activity of u-PA in fact decreased as the ras gene expression increased. These data show that the enhanced tumorigenicity of transformed murine cells is related to the tandem integration and expression of human EJ-ras. The overexpression of ras has very little effect on t-PA but appears to suppress u-PA activity.

Combined effect of pH and sodium cyanate on the inhibition of tumor cell proliferation and metabolism by BCNU and hyperthermia.

In previous studies, we have found that combined treatment with BCNU and sodium cyanate could have a greater effect on the survival of mice bearing B16 melanoma than treatment with either agent alone. With rat hepatoma and human colon cancer cells in culture, we have obtained evidence that the inhibition of cell proliferation by sodium cyanate is greater at pH 6.6 than at pH 7.4. In the present work, the effects of combination treatments on the proliferation of cancer cells were studied with cyanate, pH, BCNU, and hyperthermia. With HT29 human colon cancer cells, the inhibitory effect of BCNU (50-100 micrograms/ml) was greater when the cells were treated at pH 6.6 than at pH 7.4. The influence of pH appeared to be absent or minimal at lower or higher concentrations of BCNU. We confirmed our previous observation that the inhibition of proliferation of LS174T human colon cancer cells is greater at pH 6.6 than at pH 7.4, and we observed an inhibitory effect of BCNU (50 or 200 micrograms/ml). However, no more than additive effects were seen with combination treatment. An inhibitory effect of hyperthermia was seen for the incorporation of [3H]-leucine into protein of rat hepatoma cells (HTC) and for that of [3H]-thymidine into DNA of human colon cancer (HT29) cells. In neither case was the effect of hyperthermia significantly enhanced by treatment with sodium cyanate beyond that seen with one of the treatments alone. The data confirmed that the inhibitory effect of sodium cyanate on cell proliferation can be enhanced by a low pH but did not provide evidence for synergistic effects in combination with BCNU or hyperthermia.

alpha-Difluoromethylornithine inhibits liver metastasis produced by intrasplenic injection of human tumor cells into nude mice.

The purpose of these studies was to examine metastatic potentials of a human colon tumor xenograft (T6) and three different human tumor cell lines (LS174T, HT29 and A549) using the intrasplenic-nude mouse model system (ISMS model system). A further objective was to study the activity of alpha-difluoromethyl-ornithine (DFMO) against primary and metastatic growth of the xenograft and the three cell lines. DFMO is an irreversible inhibitor of ornithine decarboxylase, a rate-limiting step in polyamine biosynthesis. Tumor burdens in the liver of nude mice were observed 6 weeks after the intrasplenic injection with LS174T and 12-14 weeks after intrasplenic injections with T6, HT29 and A549. Most of the mice developed primary tumor growth in the spleens. DFMO showed significant activity against liver metastases but had little or no activity against primary tumor growth in the spleens of the ISMS model and against s.c. growth of the xenograft. The studies demonstrated that the ISMS model system is an excellent system for studying metastatic behavior of human tumors and for studying the antimetastatic activity of experimental drugs.

Proteins excreted by primary human colon carcinoma cells (SW 480) promote spreading and growth of metastatic human colon carcinoma cells (SW 620) in serum-free medium.

The growth behavior of the two human colon tumor cell lines (SW 480, primary and SW 620, metastatic), originating from the same patient, was studied in six different serum-free media (SFM) [GF3, Chee's essential medium plus insulin, transferrin and selenium; GF3F, GF3 plus fetuin; GF4, GF3 plus linoleic acid-BSA; GF5, GF4 plus fetuin; GF5E, GF5 plus EGF; GF5T, GF5 plus triiodothyronine]. SW 480 grew in all of the SFM. In contrast, SW 620 grew only in four SFM. The cells did not grow in GF3 and GF4. When grown in SFM, SW 480 attached much more firmly to the dishes than SW 620 as determined by the time required to detach the cells with trypsin-EDTA (SW 480, greater than 20 min and SW 620, less than 5 min). It was speculated that SW 480 cells excrete proteins in SFM which influence attachment and growth of the cells. Growth behavior of SW 480 cells which did not grow in GF3, was studied using GF3 medium and SW 480 substratum dishes. SW 620 cells readily attached to the SW 480 substratum dishes and grew. Furthermore, when SW 620 cells were grown on substratum prepared from serum-supplemented medium incubated in the absence of cells (serum substratum), the cell growth was comparable to the cell growth on SW 480 substratum in GF3. Substratum from SW 480 cells and the serum substratum were compared for their components using SDS-PAGE system. The SW 480 substratum contains many more components than serum substratum. A protein band at 60 kD appears to be common in both SW 480 and serum substrata.

Interrelationship between sodium cyanate and pH in the regulation of tumor cell division.

Previous studies have suggested that the selective inhibitory effects of sodium cyanate on tumor metabolism in vivo may be related to a lower interstitial pH in tumors. In the present work, the influence of extracellular pH on the actions of sodium cyanate was studied with one rat hepatoma cell line (HTC) and two human colon tumor cell lines (HT29 and LS174T) and with rat hepatocytes to determine if the effects are accompanied by changes in intracellular pH. With some tumor cells, an inhibition of cell proliferation was observed when the cells were exposed to an acidic medium (pH 6.6). However, the LS174T line of human tumor cells divided at pH 6.6 essentially as fast as at pH 7.4. In the concentration range of 0.02-0.1 mg/ml, a greater inhibitory effect of cyanate on cell proliferation was observed at the lower pH. Intracellular pH was found to be influenced by the sodium ion concentration of the medium to a similar degree in the three tumor lines that were examined. The intracellular pH was found to be significantly affected by cyanate in rat hepatocytes and in two of the tumor cell lines (HT29 and LS174T). The data suggested that not only does extracellular pH influence the inhibitory effect of cyanate on tumor cell proliferation but also that cyanate can affect the regulation of intracellular pH in normal and neoplastic cells.

Influence of pH on the modification of thiols by carbamoylating agents and effects on glutathione levels in normal and neoplastic cells.

In previous studies, we have suggested that the selective inhibitory effect of sodium cyanate (NaOCN) on hepatoma metabolism may be due to the lower pH observed in tumors relative to normal tissues. Lower pH might enhance the action of NaOCN by increasing the formation of isocyanic acid and carbamoylation of sulfhydryl groups. In the present work, studies were conducted on the effect of pH on the carbamoylation of sulfhydryl groups. The data indicated that carbamoylation of the sulfhydryl group of glutathione by NaOCN was enhanced by decreasing the pH from 7.4 to 6.6. A less pH-dependent response was observed with organic isocyanates. However, all reactions were reversible after the pH was increased by the addition of base. Kinetic studies showed that the rate of the reaction is very rapid, a maximal effect occurring within the first 10 min. Dose-dependent modifications of cellular glutathione by NaOCN and organic isocyanates were observed in human HT29 colon tumor cells, rat HTC hepatoma cells, and rat hepatocytes. The rate of carbamoylation of the glutathione sulfhydryl group in cells was similar to that of pure glutathione (GSH). The effect of buthionine sulfoxamine on GSH levels in cells was at least as great as that of sodium cyanate, but only the latter showed inhibitory effects on macromolecular synthesis; these were very rapid, pH-dependent, and reversible in tumor cells. Our results suggest that cellular sulfhydryl group(s) other than that of GSH might be involved in the effect of NaOCN on macromolecular synthesis.

Comparison of growth and drug response of human tumor cells in serum-free and serum-supplemented media in human tumor-clonogenic assay.

A comparison was made of growth and drug-response of five human tumor cell lines (HT-29, colon carcinoma; TWI, melanoma; A-549, lung carcinoma; Panc-1, pancreatic carcinoma; and EJ, bladder carcinoma) in serum-free media (SFM) and in serum-supplemented media (SSM) using the human tumor-clonogenic assay (HTCA) system. HT-29 cells, which had the highest plating efficiency in both SFM and SSM, were used to obtain dose-response curves for four drugs (adriamycin, 5-fluorouracil, cisplatin, and BCNU) in the HTCA. Three of the drugs (adriamycin, 5-fluorouracil, and cisplatin) produced identical drug-response curves in both SFM and SSM. These results suggest that, for some chemotherapeutic agents, results comparable to those obtained with SSM in the HTCA can be achieved using SFM. Step-by-step addition of growth factors and hormones to SFM may be a useful technique to improve some of the technical and logistic problems associated with the HTCA.

pH-related effects of sodium cyanate on macromolecular synthesis and tumor cell division.

In past work, the selective effects of sodium cyanate on macromolecular synthesis in tumors have not been seen with cells in culture. We have explored the possibility that differences in the response of tumor cells to cyanate in vivo and in vitro may be related to the pH in the environment to which cells are exposed. When rat hepatoma (HTC) cells were incubated with sodium cyanate (0.25 mg/ml), there was a greater inhibition of precursor incorporation into RNA and DNA with a decrease in pH from 7.4 to 6.6. At pH 7.4 there was no significant effect of sodium cyanate on the incorporation of [3H]leucine into protein of rat hepatocytes and HTC cells, but at pH 6.6 there were decreases of 50% or greater. The time of response and the reversibility of the inhibitory effects of sodium cyanate were not those anticipated from carbamoylation of amino groups but were compatible with modification of sulfhydryl groups. The uptake of [14C]sodium cyanate in HTC cells and human colon cancer (HT29) cells was greater at pH 6.6 than at 7.4. Over a period of 4 days there was a slower rate of cell division by HTC and HT29 at pH 6.6 than at pH 7.4. The addition of sodium cyanate caused a further reduction in the rate of proliferation, and at a concentration of 0.25 mg sodium cyanate/ml there were decreases in cell numbers. The data suggested that a lower interstitial pH in tumors than normal tissues would result in greater sensitivity to inhibitory effects of sodium cyanate on macromolecular synthesis.

Biochemical analysis of the effects of 1,3-bis(2-chloroethyl)-1-nitrosourea on the growth of a human melanoma cell line.

A study of the biochemical effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on the growth of human melanoma cells (TWI) revealed that this antitumor agent inhibits: (i) the synthesis of ribosomal RNA, (ii) the synthesis of cellular proteins of molecular weight greater than 49 kDa and (iii) the synthesis and release of extracellular proteins to the growth medium. [3H]Uridine labelling of RNA species coupled with agarose gel electrophoresis analysis and fluorography gave a nicely resolved picture of the sequence of events in control and treated cells. Protein analyses were done using polyacrylamide gel electrophoresis.