Degradation of vascular subendothelium occurs in_vivo during the process of inflammation and tumor invasion. Various observations suggest that the capacity of some blood-borne cells to extravasate may depend in part on their ability to express hepara-nase activity. Incubation of human platelets, human nc-utrophils, or highly metastatic mouse lymphoma cells with sulfate-labeled extracellular matrix (ECM) results in heparanase mediated release of labeled heparan sulfate cleavage fragments (0.5<Kav<0.85 on Sepharose 5B) (J. Clin.Invest. 74: 1842 and 76: 1306; Cancer Res. 43: 2704). The present study was undertaken to test the heparanase inhibitory effect of heparin and non-anticoagulant species of heparin that might havea potential therapeutic use in preventing heparanase mediated extravasation ofblood-borne cells. We prepared totallyor N-desulfated heparins which were either left with their N-position exposed or were subsequently N-acetylated or N-resulfated. These heparins exhibited less than 5% of the anticoagulant activityof native heparin. It was found that total desulfation of heparin abolished its heparanase inhibitory activity whether desulfation was followed by N-acetylation or not. Inhibitory effect was restored by resulfation of the N-position. When only the N-sulfate group was desulfated, inhibitory activity was lost but could be restored by acetylation of the N-position. These results indicate that N-sulfate groups of heparin are necessary for its heparanase inhibitory activity but can be substituted by an acetyl group provided that the 0-sulfate groups are retained. Low Mr heparins (main Mr species of 2500 and 4500 daltons) and heparin fragments as small as the tetrasaccharide inhibited degradation of heparan sulfate in the ECM, albeit to a lower extent than native heparin. Similar effects of the different heparins were observed with heparanase activities from platelets, neutrophils and lymphoma cells. Preliminary in vivo experiments suggest that non-anticoagulant heparins interfere with tumor metastasis and experimental autoimmune diseases (some heparins were kindly provided by Inst. Choay, Paris and Kabi Vitrum, Stockholm).
Abstract Renal cell carcinoma is a chemotherapy-resistant tumor which is relatively responsive to immunotherapy. Immunotherapeutic regimes employ interferons or interleukin 2 with or without lymphokine-activated killer cells. Secondary cytokines, induced by interleukin 2 or interferon, may have an important impact on their anti-neoplastic activity. Notable among them is tumor necrosis factor (TNFα). We assessed the effect of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) on the susceptibility of the human renal cell carcinoma cell line SK-RC-29 to the cytotoxic and cytostatic actions of TNFα, interferon a and lymphokine-activated killer cells. Using uptake of the vital dye neutral red as an indicator of viable cell number, we found that addition of 1,25(OH) 2 D 3 (100 n m ) to TNFα (30 ng/ml)-treated cultures resulted in a 2·6 ± 0·2-fold (mean ± s.e. ) increase in the cytotoxic effect of the cytokine. The potentiating effect of 1,25(OH) 2 D 3 was dose-dependent, and significant at concentrations equal to or higher than 10 n m . Another dihydroxylated vitamin D metabolite, 24,25(OH) 2 D 3 , had no effect on TNFα action. The cytotoxic effect of TNFα increased whereas the potentiation by 1,25(OH) 2 D 3 decreased with cell density in culture. 1,25(OH) 2 D 3 , in contrast to its potentiating effect on TNFα action, did not modulate the cytostatic effect of interferon α or the susceptibility of SK-RC-29 to killing by lymphokine-activated killer cells. The findings reported here may explain some of the in vivo anti-tumor activity of 1,25(OH) 2 D 3 and provide a rationale for the employment of active vitamin D analogs during immune anti-cancer therapy. Journal of Endocrinology (1996) 149, 327–333
