Therapeutic response of human tumor xenografts in athymic mice to doxorubicin.

In order to establish the usefulness of the human tumor-nude mouse system as a predictive screen for anticancer agents, 17 tumors (3 breast, 3 colon, 3 lung, 3 melanoma, 2 ovary, 1 prostate, 1 sarcoma, and 1 larynx), serially transplantable in athymic mice, were used to study antitumor activity of doxorubicin (Adriamycin). BALB/c nude mice were treated i.v. on a weekly basis for 3 to 4 weeks, starting when the tumor volume became relatively large (advanced stage of tumor treatment). All the tumors except lung tumor T 293 showed a 90 to 100% take rate and stable growth. Doxorubicin, at dose levels of 6 and 10 mg/kg/injection i.v. every week for 3 weeks, showed significant activity against all of the three breast tumors studied. As was expected on the basis of clinical data, doxorubicin showed no antitumor activity against the three different colon tumors. In the case of lung tumors, statistically significant activities against oat cell carcinoma T 293 and epidermoid carcinoma T 222 were observed. In contradiction to clinical data, doxorubicin was found to have significant activity against various melanomas studied and slight but not statistically significant activity against ovarian tumor T 17. Experimental results obtained using doxorubicin against prostate, sarcoma, and larynx tumors also parallel the reported clinical data.

Growth state-specific responsiveness of primary cultures of a nude mouse-xenografted human colon carcinoma to 4'-deoxydoxorubicin and a crude human leukocyte alpha-interferon preparation.

Improved procedures for the seeding of primary cultures from human colon tumors are described. Responsiveness of primary cultures from human colon carcinoma T348 to a crude preparation of human leukocyte alpha-interferon and a DNA-intercalating experimental anticancer drug, 4'-deoxydoxorubicin, has been investigated. The effect of the interferon preparation on such cultures is shown to be growth state specific; i.e., stationary populations are killed, whereas fast-growing cultures are reversibly growth inhibited by the same doses of interferon. In contrast, 4'-deoxydoxorubicin kills growing populations, whereas stationary cultures are barely affected by the same concentration of this drug. Interferon antagonizes the cytotoxic effect of 4'-deoxydoxorubicin on a growing colon tumor cell population when applied immediately after 4'-deoxydoxorubicin treatment. Implications of this finding for the combined application of several drugs in cancer therapy are discussed.

In vitro responses of nude mouse-xenografted human colon carcinomas exposed to doxorubicin derivatives in tissue culture and in the mouse.

In vitro responses of 4 xenografted human colon tumors (T183, T219, T245, and T348) to various doses of 4'-deoxydoxorubicin have been investigated. The individual tumors showed marked differences in drug responsiveness, ranging from high sensitivity at low doses (T219; 125 ng/ml) to very low sensitivity at high doses (T245; 4000 ng/ml). The sensitivity ranking deduced from these in vitro experiments correlates well with the ranking deduced earlie (Guiliani et al., Int. J. Cancer, 27: 5-13, 1981) from in vivo drug treatments of transplants of these tumors in the nude mouse. The effect of in vitro drug treatment (4'-deoxydoxorubicin; 250 ng/ml; 1-hr incubation) on the in vivo growth of one of the tumors, T219, in nude mice was investigated. Growth of the tumor in nude mice was markedly delayed by pretreatments in vitro with 4'-deoxydoxorubicin. Furthermore, in vitro responsiveness of the T219 tumor was investigated following in vivo and in vitro treatment of the tumor with 4'-deoxydoxorubicin. Both of the pretreatments produced very similar decreases in drug responsiveness to all of the doxorubicin derivatives tested (4'-deoxydoxorubicin, 4'-O-methyldoxorubicin, 4'-epidoxorubicin, 4-demethoxydoxorubicin, and N-trifluoroacetyldoxorubicin-14-valeriate).

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.

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.

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.

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.

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.

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.

Activity of cancer chemotherapeutic agents against human colorectal carcinomas grown as primary tissue culture.

Improved procedures are described for the seeding of primary cultures from human colon adenocarcinoma and for the use of these cultures in the evaluation of drug effects. Two of the specimens studied were xenografts maintained in athymic (nude) mice, while the other six were biopsies obtained directly from patients. Tumor cells obtained directly from the patients proliferated in defined hormone-supplemented medium to the exclusion of other cells. In drug-response studies with cultures from a colon tumor biopsy all four drugs studied (4'-deoxydoxorubicin, 4'-O-methyldoxorubicin, 5-fluorouracil, and 1,3-bis-[chloroethyl]-1-nitrosourea) inhibited growth of the cells within 3-6 days after drug treatment. On an equitoxic dose basis (LD10 in mice), 4'-deoxydoxorubicin appeared to be the most active drug. This drug also showed dose-dependent activity against one of the xenografted tumors in vitro. In dose-response studies with cultures from another patient's colon tumor, doxorubicin and 5-fluorouracil showed significant activity against the tumor 10 days after the drug treatment with concentrations at 1X and 10X average peak plasma levels.

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.

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.

Biochemorphology of cyclobutanecarbonylureas.

Ten urea derivatives of cyclobutanecarboxylic acid were synthesized and examined for general CNS depressant properties, barbiturate potentiation, and myorelaxant, antitremorine, and anticonvulsant potencies. Although water solubility plays an important role in the activity of these compounds, other factors also appear to be involved. 1-Cyclobutanecarbonyl-3,3-dimethylurea appears to be the most active CNS depressant, whereas 1-cyclobutanecarbonyl-3-(alpha-naphthyl)thiourea is the most active barbiturate potentiator. 1-Cyclobutanecarbonyl-3,3-dimethylurea, 1-cyclobutanecarbonyl-3-phenylurea, and 1-cyclobutanecarbonyl-3-tert-butylurea, 1-cyclobutanecarbonyl-3-allylurea, and 1-cyclobutanecarbonyl-3-phenylurea apparently are the most active against pentylenetetrazol-induced convulsions. 1-Cyclobutanecarbonyl-3-tert-butylurea, 1-cyclobutanecarbonyl-3,3-dimethylurea, and 1-cyclobutanecarbonyl-3-phenylurea are also slightly active tremorine antagonists.

Sinoacutine from Glaucium contortuplicatum Boiss.

A phytochemical investigation of Glaucium contortuplicatum Boiss. (Papaveraceae) resulted in the isolation of sinoacutine from this plant for the first time. Spectral evidence for the identity of the isolated compound as sinoacutine is presented.

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.