Impact of antioxidants on the ability of phenolic phytochemicals to kill HCT116 colon cancer cells.

Certain phenolic phytochemicals can kill cancer cells. Possible interference from antioxidants is a concern, and this issue has not been studied appreciably. Therefore, the effect of ascorbate and N-acetylcysteine on the ability of epigallocatechin gallate (EGCG) and curcumin to kill HCT116 colon cancer cells was examined. EGCG and curcumin each caused DNA damage in the cells. The DNA-damaging ability of EGCG, but not curcumin, was hindered by either ascorbate or NAC, which was also shown in HT29 and SW480 colon cancer cells. Also, iron chelators (deferoxamine and 2,2'-dipyridyl) inhibited the ability of EGCG, but not curcumin, to cause damage to the DNA in HCT116 cells. Interestingly, curcumin, but not EGCG, increased the expression of growth arrest and DNA damage-inducible gene 153 and also heme oxygenase-1, and this stress gene upregulation by curcumin was antioxidant-insensitive. With prolonged incubation of HCT116 cells with either EGCG or curcumin, cell shrinkage, membrane blebbing, apoptotic bodies, and chromatin condensation/fragmentation were observed. These morphological changes were not apparent in EGCG-treated cells that had been pretreated with either ascorbate or NAC. However, the ascorbate and NAC pretreatments did not prevent the occurrence of the morphological changes in curcumin-treated cells. Thus, these findings suggest that ascorbate and NAC interfere with the ability of EGCG, but not curcumin, to kill HCT116 cells. This basic knowledge may help to better plan and optimize strategies for chemoprevention or chemotherapy.

Evaluation of premalignant and malignant lesions during the induction of mouse melanomas.

The objective of the present study was to evaluate the malignant and premalignant lesions that arise in C57BL/6 mice after treatment with 7,12-dimethylbenz(a)anthracene and croton oil. Tissues from 70 treated mice were evaluated by histological and transplantation techniques, and 17 (24%) were found to have malignant tumors. Eleven of the tumors were diagnosed as malignant melanomas, three as spindle cell sarcomas, and three as squamous cell carcinomas. The incidence of malignant melanomas (15.7%) in this group of mice was similar to that in our initial study on the induction of melanomas with 7,12-dimethylbenz(a)-anthracene and croton oil, in which two of 20 mice developed malignant melanomas. Mice that developed melanomas had been treated with croton oil for an average of 7 mo, and the mean latent period for tumor development was 11 mo. Seven of eight melanomas grew rapidly after transplantation to syngeneic C57BL/6 mice. Pigmented nevi and/or draining lymph nodes from nine of 11 mice with melanomas grew progressively after transplantation to athymic nude mice. Pooled nevi from one mouse with no apparent tumors grew into a histologically malignant melanoma after transplantation to a nude mouse. Nevi from three mice with sarcomas, one mouse with a carcinoma, and 42 tumor-free treated mice failed to grow in nude transplant recipients. Thus, only nevi from mice with either apparent or occult malignant melanomas exhibited progressive growth in nude mice. These results confirm that malignant melanomas can be induced in C57BL/6 mice at a regular, predictable rate and further indicate that this is an excellent system in which to study melanoma induction, progression, and therapy.

Phenotypic instability of mouse melanomas after propagation in vivo and in vitro.

This study was designed to evaluate the phenotypic stability of the carcinogen-induced JB/MS and JB/RH melanomas. The JB/MS melanoma maintained its original slow-growing melanotic phenotype during in vivo passage over a 2-yr period. However, both melanin production and tumorigenicity decreased rapidly after propagation of JB/MS cells in vitro. The JB/RH melanoma became essentially amelanotic after the second transplantation in vivo. Cultured JB/RH cells produced tumors identical to those obtained by transplantation of JB/RH tumor fragments. However, after propagation in vitro for 2 yr, the JB/RH cell line decreased in tumorigenicity, requiring 10 times as many cells to produce tumors in C57BL/6 mice as did the original cell line. The JB/RH melanoma was highly immunogenic in syngeneic C57BL/6 mice, and passage of JB/RH cells through immunized mice resulted in tumors that were significantly more tumorigenic in normal mice than were JB/RH cells that had been passed through either nude or sublethally irradiated mice. These results indicate that, in studies of primary mouse melanomas, it is essential to: (a) limit the number of tumor passages; (b) choose methods of propagation that will preserve the original phenotype; and (c) distinguish those properties produced by technical manipulation from those produced by true tumor progression.

Uveitis caused by cytotoxic immune response to cutaneous malignant melanoma in swine: destruction of uveal melanocytes during tumor regression.

In a substrain of Sinclair miniature black swine, bred for increasing incidence of cutaneous malignant melanomas, tumor regression occurs spontaneously and is accompanied by depigmentation of the skin, hair, and eyes. We conducted a 12-month longitudinal study of the ocular phenomena in 30 swine beginning at 3 weeks of age. The clinically observed sequence of depigmentation of the fundus and iris was correlated with histopathologic changes in selected enucleated eyes. Normal melanocytes of the uveal tract are destroyed between the 4th and 16th week of life. Melanocyte destruction is preceded by an invasion of the uveal tract by mononuclear cells having the ultrastructural features of lymphocytes and monocytes. Melanin and other cellular debris of ruptured melanocytes are ingested by macrophages which then migrate to the walls of blood vessels. Cataracts and band keratopathy develop secondary to the uveitis in some animals. Pilot electroretinograms show diminished electrical activity in photoreceptors of totally depigmented eyes possibly indicating ischemic or toxic damage to the retina. The retinal pigment epithelium remains essentially normal during the acute stages of uveal inflammation; later some damage and reparative hyperplasia may occur. The death of normal uveal melanocytes that occurs during the systemic attack on the cutaneous malignant melanomas appears to be an "innocent bystander" error in the immune recognition mechanism. The antigenic basis of this immunologic cross reaction is under investigation.

Cell-mediated immune reactivity of Sinclair melanoma-bearing swine to 3 M KCl extracts of swine and human melanoma.

Leukocyte migration inhibition (LMI) assays were performed using 3 M KCI extracts to detect cell-mediated immune reactions against tumor-associated antigens (TAA) of swine melanoma. Positive LMI reactivity was 70% or greater in melanoma-bearing swine compared to 10% reactivity or less in normal swine tested with extracts of either fresh or tissue-cultured swine melanoma cells. Positive LMI reactivity was 10% or less in both melanoma and normal swine tested with extracts of normal fetal and adult swine tissues. Extracts of tissue-cultured swine melanoma and human melanoma cells were equally capable of stimulating positive LMI reactivity in melanoma swine and human melanoma patients; normal swine and human donors demonstrated 10% or less positive LMI reactivity to these extracts. Positive LMI reactivity was not stimulated in swine melanoma or human melanoma donors by extracts of tissue-cultured human breast tumor cells or fresh and tissue-cultured mouse B16 melanoma cells. Evaluation of these results indicate that melanoma-bearing swine develop cell-mediated immune reactivity toward TAA on swine melanomas and, inasmuch as LMI assays were performed with allogeneic extracts, suggests the presence of common TAA on swine melanomas. Furthermore, these data suggest that swine and human melanoma cells may share some common TAA and that this common TAA is not expressed on mouse B16 melanoma cells.

Growth and regression of cutaneous melanomas in Sinclair miniature swine.

Previous studies have demonstrated pathologic similarities between human melanoma and the spontaneous melanoma in the Sinclair miniature swine (SMS) with respect to cutaneous histologic features and patterns of metastasis. The current biopsy series, correlating growth curves and histopathologic features of cutaneous melanomas, was undertaken for documentation of the histologic events associated with the successful regression of melanoma in the SMS. Cutaneous growth and regression was characterized by a series of cellular events that eventually led to depigmentation and scar formation. Mononuclear inflammatory infiltrates, seen in over half of the 104 biopsies, showed several temporal and topographic distribution patterns, similar to that described in human melanoma. Histopathologic observations in the SMS confirm clinical observations that the host can, with consistent effectiveness, react with the tumors to modify their biologic aggressiveness. Although regression is associated with lymphocytic and macrophage infiltration, the exact role of the immune response in the regression of the cutaneous melanoma remains to be elucidated.

Development of a new melanoma model in C57BL/6 mice.

In the present study, we induced melanomas in C57BL/6 mice by a single application of 7,12-dimethylbenz(a)anthracene to the scapular region of 4-day-old mice, followed by twice-weekly applications of croton oil. Of 20 mice treated, melanomas arose in two female littermates. The first melanoma (JB/MS) arose 16 weeks after initiation of treatment, and the second melanoma (JB/RH) arose 23 weeks later. The melanomas maintained their melanotic appearance after s.c. transplantation to normal C57BL/6 mice and metastasized spontaneously in the transplant recipients. To our knowledge, these are the first melanomas to have been induced in C57BL/6 mice since the B16 melanoma arose spontaneously in 1954. We feel that the JB/MS and JB/RH melanomas provide an excellent comparative system for studies done with the B16 melanoma. These melanomas of recent origin will also facilitate the investigation of biological, immunological, and biochemical parameters that influence the growth and metastasis of malignant melanomas.

Growth and spontaneous regression of swine melanoma: relationship of in vitro leukocyte reactivity.

A visual microcytotoxicity technique was used to evaluate the leukocyte reactivity of melanoma swine against allogeneic swine melanoma target cells. Peripheral blood leukocytes, which had been collected and cryopreserved in liquid nitrogen at various times during in vivo tumor growth and regression, were thawed and tested on the same day. Comparison of longitudinal leukocyte reactivity with in vivo tumor volume indicated that swine with regressing melanomas exhibited increased leukocyte reactivity during tumor regression. Swine with maximum tumor volumes less than 30,000 mm3 exhibited patterns of leukocyte reactivity that paralleled the patterns of in vivo tumor growth and regression. However, swine with maximum tumor growth and regression. However, swine with maximum tumor volumes greater than 30,000 mm3 demonstrated increased in vitro leukocyte reactivity at the time of maximum in vivo tumor volume. Histopathologic analyses revealed that increases in tumor volume were frequently a result of host inflammatory cells, particularly pigment-laden macrophages, infiltrating the tumors. Thus at the time of maximum tumor volume, malignant melanocytes were proportionately decreasing in number while pigment-laden macrophages were proportionately increasing. These studies provide additional evidence that the spontaneous tumor cell regression of swine melanoma is associated with immunologic events and that assays of leukocyte reactivity are useful in vitro correlates of host antitumor immunity.

Growth of Sinclair swine melanoma in the hamster cheek pouch.

Specimens from seven Sinclair swine melanomas were transplanted to the cheek pouches of Syrian Golden hamsters. The specimens were taken from young swine and were derived from raised tumors that either were present at birth or developed after birth from flat melanocytic lesions as well as from apparently normal skin. All seven specimens grew in the hamster cheek pouch. One lesion, derived from a 3-day-old piglet, exhibited the most aggressive growth in the hamster and was successfully transferred to other hamster cheek pouches. These results confirm the malignancy of Sinclair swine melanoma and indicate that tumors of neonatal swine contain more malignant cells than those of older animals.

Adaptation of Sinclair swine melanoma cells to long-term growth in vitro.

Sinclair swine melanoma usually regresses in vivo. In the present study, swine melanoma cells were adapted to long-term growth in culture. The morphology of cultured melanoma cells ranged from dendritic to cuboidal, similar to that described for human melanoma cells. Doubling times of the swine melanoma cells were also similar to those of human melanoma cells in vitro. 3,4-Dihydroxy-L-phenylalanine oxidase-positive cells were detected by light microscopy, and melanin and premelanosomes were detected by electron microscopy. Cell cultures could be propagated from progressing, partially regressed, and primary cutaneous lesions, as well as from visceral metastases. Thus, it appears that, under these cell culture conditions, Sinclair swine melanoma cells can be adapted to prolonged growth in vitro.

A clinical histologic, and immunologic study of a case of metastatic malignant melanoma undergoing spontaneous remission.

A patient with biopsy-proven dermal recurrent malignant melanoma who refused therapy, and who was observed to undergo clinical regression during the period of November 1972 through June 1974 was studied to define the histologic features of spontaneous remission, and to evaluate the immune response as measured by in vitro assays of lymphocyte cytotoxicity and serum effects during the course of regression. Biopsy of regressed areas showed the following histologic features: 1) absence of malignant melanoma cells in basal layers of epidermis with relative increase in basal layer clear cells; 2) dermal inflammatory reaction with lymphocytic infiltrate, melanophages, and degenerate malignant melanocytes; and 3) dermal reactive vascular proliferation and interstitial edema progressing to reparative dermal fibrosis. Using a microcytotoxicity assay with two established allogeneic melanoma cell cultures as target cells, a statistically significant (p less than 0.01) increase in lymphocyte cytotoxicity values was observed over the clinical time course of regression. No significant serum cytotoxic or serum blocking effects were detectable. These findings are consistent with an immunologic basis for the spontaneous remission of the dermal melanoma metastases present in this patient.

Regression of pulmonary metastatic disease associated with intralesional BCG therapy of intracutaneous melanoma metastases.

A 77-year-old white man with 64 intracutaneous melanoma metastases and a pulmonary metastatic deposit was treated with immunotherapy. Over an 8-month period, 17 intracutaneous lesions were inoculated with BCG. All 17 injected lesions and all 47 uninjected intracutaneous lesions resolved; no new nodules appeared and the pulmonary metastasis regressed (greater than 50%). This is the first documented case of a pulmonary metastatic focus responding to intralesional BCG therapy of intracutaneous metastases.

Sequential in vitro reactivity of lymphocytes from melanoma patients receiving immunotherapy compared with the reactivity of lymphocytes from healthy donors.

A microcytotoxicity technique was used to determine the sequential in vitro reactivity against melanoma cells of lymphocytes from melanoma patients receiving immunotherapy and from healthy donors. Lymphocytes were collected 2 weeks for 2-3 months and were stored in liquid nitrogen until use. Preliminary studies had indicated that freezing did not effect the reactivity of lymphocytes. Lymphocytes from 10 healthy donors tested against melanoma cells exhibited substantial reactivity which showed no consistent pattern over time. Lymphocytes from 9 melanoma patients exhibited increased reactivity after immunotherapy. Patterns of reactivity against melanoma cells and against bladder carcinoma cells were similar, indicating lack of specificity for melanoma antigens. Correlations with clinical course of the disease were not apparent.