Detection of glucocorticoid sensitive secretory proteins from human melanoma cells

NEL-M1 human melanoma cells have been established to grow in Ham's F12 medium in the absence of serum, hormones, and exogenous growth factors. Conditioned medium from NEL-M1 cultures stimulates growth of these same cells whereas glucocorticoids retard growth in the presence and absence of conditioned medium. Because recent reports indicate that glucocorticoids inhibit the synthesis of growth factors from a variety of cell types, we hypothesized that glucocorticoids may be inhibiting growth of NEL-M1 cells by either suppressing the synthesis of the autocrine growth factor or regulating other secretory proteins that may inhibit the activity of the autocrine growth factor. Initial studies were done to clearly show that NEL-M1 cells were growth inhibited, both in vivo and in vitro, when exposed to the synthetic glucocorticoid, triamcinolone acetonide (TA). When NEL-M1 cells were injected into nude mice and treated with TA (100 micrograms per mouse per week) a 67% reduction in tumor mass was observed compared to the control group over a 5-wk growth period. Additional studies show that in a serum-free defined medium TA (100 nM) inhibited growth of NEL-M1 cells by 56% after 6 d in culture. At this same time TA was shown to affect the expression of several proteins secreted from these cells. TA treatment resulted in the appearance of a 125,000 molecular weight protein, suppression of the synthesis-secretion of three proteins (37,000, 57,000, and 76,000 molecular weight) and enhanced expression of a 60,000-molecular weight protein. However when NEL-M1 cells were cultured in conditioned medium from TA-treated cells, a stimulation in both [3H]thymidine incorporation into DNA and cell proliferation was observed. When the conditioned medium was fractionated by Amicon ultrafiltration, the growth stimulatory activity was found in the less than 10,000 molecular weight fraction. These results demonstrate that glucocorticoids, as a single agent, inhibit the growth of NEL-M1 human melanoma cells. However, this growth inhibition by glucocorticoids may not be through the regulation of the synthesis-secretion of the autocrine growth factor. Furthermore, the data suggest that the glucocorticoid-sensitive secretory proteins may not be directly involved in the in vitro regulation of NEL-M1 cellular growth.