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Tumor cells isolated from human renal cell carcinoma biopsies were treated with cholesteryl hemisuccinate or 25-hydroxycholesterol and the subsequent changes in their membrane fluidity and capacity to induce skin reactivity in the homologous patient were investigated. Both cholesterol derivatives were found equally efficient in decreasing membrane fluidity when measured by fluorescence polarization of diphenylhexatriene. Using trimethylammonium-diphenylhexatriene, a specific cell surface probe, 25-hydroxycholesterol, appeared much more efficient than cholesteryl hemisuccinate in inducing a membrane rigidification in the carcinoma cells. Cells treated with cholesteryl hemisuccinate induced a strong positive skin reaction compatible with delayed-type hypersensitivity in 54% of the patients, whereas 25-hydroxycholesterol-treated cells were less potent (36% positive skin reactions). Thus, manipulation of the physicochemical state of the membrane of human renal carcinoma cells could increase their immunogenicity in the autologous patient, although this seemed not to be related only to membrane rigidification.

The antitumor effects of two new hydrosoluble derivatives of oxygenated sterols: JB69 and JC40 have been evaluated in vitro on a panel of lymphoma and leukemia cells from human and murine origins. These compounds result from the combination of a nucleotide with 7 beta-hydroxycholesterol (JB69) or 7 beta,25-dihydroxycholesterol (JC40). Both derivatives exhibit a significant cytotoxic activity against the different tumor cell lines tested, with some degree of difference between them. On the whole, the concentrations needed to inhibit the cell growth were found to be higher than those required for their parent compounds. However, two interesting features appeared in our experiments. (1) In a serum-free culture medium, cell lysis occurred within the first hours of incubation and seemed to result from the detergent-like properties possessed by this type of compounds. (2) In a culture medium supplemented with serum, we noted, that at high concentrations of JB69 (40 microM or 20 microM) and only with this oxysterol derivative, an important increase of incorporation of tritiated thymidine and uridine into DNA and RNA by viable cells. The origin of this effect is as yet unknown, but it strongly suggests a possible action on nucleic acids synthesis and metabolism. Taken together, these results emphasize the diversity and the complexity of the mechanisms involved in the cytotoxicity of these derivatives of oxysterols.

It was hypothesized that membrane rigidification of tumor cells could enhance the expression of tumor associated antigens (6) For this purpose, we treated in vitro hypernephroma cell with cholesterol hemisuccinate (CHS) or 25-hydroxysterol (250HC) and we evaluated their immunogenicity by skin-tests in 26 patients after radical nephrectomy. The skin-tests were positive in 50% cases with CHS treated cells, and in 35% with 250HC treated cells. The immune responses were characterized as delayed hypersensitivity. However determinations of all membrane fluidity suggested that membrane rigidification was not the unique factor involved. These results could therefore be of clinical interest in the treatment of renal carcinoma by active immunotherapy.

The cytostatic and cytolytic action of 22R - hydroxydesmosterol on hepatoma cells cultured in a medium containing 10% newborn-calf serum can be reversed within certain concentration limits by adding cholesterol to the culture medium. In contrast, under the same conditions, the cytotoxicity of 7 beta -hydroxycholesterol could not be reversed, whatever the concentrations of cholesterol added. However, in a lipoprotein-poor and in a chemically defined medium, the cytolytic action of both hydroxysterols can be reversed by adding cholesterol, but growth inhibition cannot be suppressed. This demonstrates the importance of serum lipids and lipoproteins for the toxicity of the hydroxysterols and for the antagonistic effect of cholesterol. Our results suggest that the action mechanisms of 7 beta-hydroxycholesterol and 22R - hydroxydesmosterol on HTC hepatoma cells are not fully identical.

When murine BALB/c splenocytes are pretreated for 2 h with different concentrations of 25-hydroxycholesterol (25-HC) or 7 beta-hydroxycholesterol (7-HC), washed and stimulated either with irradiated C57BL/6 splenocytes or with Con A, IL-2 secretion is inhibited in a dose-dependent way as well as the subsequent cell proliferation. Using the same treatment and stimulation conditions, IL-2 receptor appearance on human T lymphocytes, as characterized by anti-Tac antibody binding, is also inhibited in a dose-dependent way. In contrast, the hydrophilic derivative of 7 beta-hydroxycholesterol, the 3.7 bishemisuccinate sodium salt (7-HC BHS), did not influence any of the 3 tested parameters.

Several oxysterols are powerful immunosuppressors. Among them 7,25-dihydroxycholesterol (7,25-OHC) seems to be particularly interesting. The effect of this synthetic sterol on murine lymphocytes, stimulated by Con A, or by allogeneic or TNP-modified syngeneic lymphocytes, was investigated. The results showed that the induction of cytotoxic T-lymphocytes (CTL) was strongly inhibited by a treatment with non-toxic concentrations of this sterol. A marked reduction of interleukin 2 (IL-2) production by alloantigen stimulated lymphocytes occurred at the same concentrations. We also showed that receptors to IL-2 did not appear on lymphocytes stimulated by Con A, when 7,25-OHC was added at the beginning of the culture. The addition of exogenous IL-2 to the culture was found to only slightly reverse the inhibition caused by 7,25-OHC. Taken together, these results indicate that an impairment at the early steps of lymphocyte activation, involving IL-2 secretion, could be responsible, at least in part for the various immunosuppressive effects of 7,25-OHC.

Sodium bis-hemisuccinates of 7 beta- and 7 alpha-hydroxycholesterols are moderately water-soluble. They have been tested intraperitoneally against the murine Krebs-II carcinoma, grown as an ascitic tumour, and their action has been compared with that of usual chemotherapeutic drugs, cyclophosphamide, 5-fluoro-uracil, and methotrexate. The hydroxycholesterol derivatives show a faster and stronger activity (life prolongation), and lead to the complete disappearance of the tumour in about 1/3 of the cases, even with one single injection. Similar results have been obtained (on fewer cases) with two other experimental ascitic tumours, the S-180 sarcoma and the ZHC hepatoma. The mechanism of action is not known; it appears to be very different from that of the usual anti-cancer chemotherapeutic agents.

The effects of 7 beta-hydroxycholesterol on lymphoma cells in culture, on lymphocytes entering blastic transformation and on quiescent murine spleen lymphocytes have been investigated. The early events of blastogenesis as well as YAC-1, RDM-4 and EL-4 cells were shown to be very sensitive to this sterol at microM concentration, whereas constituted lymphoblasts and normal lymphocytes remained insensitive at 50 times higher concentration. The effect of some classical antitumor drugs (Adriamycin, Mitomycin-C, Methotrexate) on these lymphoma cells were of the same order of magnitude. However, the activity of 7 beta-hydroxycholesterol was closely related to the composition of the culture medium. Indeed, the cytotoxic effects of this compound were less in medium supplemented with foetal calf serum than in lipoprotein poor, Ultroser-G supplemented medium. The possible impairment of the same, or closely related, events occurring in blastic transformation and in rapid proliferation of cells is pointed out. Our results raise the question of the possible use of these compounds for an antitumor strategy.

The toxicity of 7 beta-hydroxycholesterol for cultured HTC cells is 10 times greater if serum lipids and lipoproteins are absent from the culture medium. A water-soluble derivative of 7 beta-hydroxycholesterol, sodium 3,7-bishemisuccinate, showed the same toxicity as the original molecule and was also 8 times more toxic when serum lipids and lipoproteins were absent. But the rapid inhibition of DNA synthesis was similar in cells treated with both compounds, whether lipids and lipoproteins are present or not. Thus the absence of serum lipids and lipoproteins enhances the lytic effect of both substances but does not increase their intracellular action on DNA synthesis. This first parallel study on lipophilic 7 beta-hydroxycholesterol and its water-soluble homologue shows the importance of the serum lipids and lipoproteins in the cytotoxicity of such sterols.