Receptor-mediated uptake of low-density lipoprotein by B16 melanoma cells in vitro and in vivo in mice

Abstract

Summary Selective delivery of cytotoxic anti-neoplastic drugs can diminish the severe side-effects associated with these drugs. Many malignant tumours express high levels of low-density lipoprotein (LDL) receptors on their membranes. Therefore, LDL may be used as a carrier to obtain selective delivery of anti-neoplastic drugs to tumours. The present study was performed to investigate the feasibility of the murine B16 tumour/mouse model for the evaluation of LDL-mediated tumour therapy. LDL binds with high affinity to LDL receptors on cultured B16 cells (Kd, 5.9+2.3 Yg ml -; Bmax, 206+23 ng LDL mg-1 cell protein). After binding and internalisation, LDL was very efficiently degraded: 724+ 19 ng LDL mg -cell protein h-'. Chloroquine and ammonium chloride completely inhibited the degradation of LDL by the B16 cells, indicating involvement of lysosomes. LDL receptors were down-regulated by 70% after preincubation of B16 cells with 300 jg ml-l LDL, indicating that their expression is regulated by intracellular cholesterol. To evaluate the uptake of LDL by the B16 tumour in vivo, tissue distribution studies were performed in C57/Bl mice inoculated with B16 tumours. For these experiments, LDL was radiolabelled with tyramine cellobiose, a non-degradable label, which is retained in cells after uptake. At 24 h after injection of LDL, the liver, adrenals and the spleen were found to be the major organs involved in LDL uptake, with tissue-serum (T/S) ratios of 0.82+0.08, 1.17 + 0.20 and 0.69 + 0.08 respectively. Of all the other tissues, the tumour showed the highest uptake of LDL (T/S ratio of 0.40+0.07). A large part of the LDL uptake was receptor mediated, as the uptake of methylated LDL was much lower. Although the LDL uptake by the liver, spleen and adrenals is higher than that by the tumour, the LDL receptor-mediated uptake by these organs may be selectively down-regulated by methods that do not affect the expression of LDL receptors on tumour cells. It is concluded that the B16 tumour-bearing mouse constitutes a good model to evaluate the effectiveness of LDL-mediated delivery of cytotoxic (pro)drugs to tumours in vivo.

Low-density lipoprotein receptor-mediated delivery of a lipophilic daunorubicin derivative to B16 tumours in mice using apolipoprotein E-enriched liposomes

“…earlier results indicate that LDL is processed by the B 16 tumour inoculated in mice. albeit at a somewhat lower rate than in liver and adrenals (Versluis et al 1996). The tissue distribution of LAD-loaded apoE-liposomes is very similar to that of native LDL.…”

“…1974). We showed recentlI that the interaction of LDL with B 16 cells is also strongly Ca'+ dependent (Versluis et al 1996). Percentage of injected dose g-' Figure 6 Tissue distribution of LAD-loaded apoE-liposomes.…”

“…For in vitro experiments, LDL was labelled using the ['5l]iodine monochloride method as described in detail by Bilheimer et al (1972). For in vivo experiments, LDL was labelled with '1I-tyrammne cellobiose ('-I-TC) as described previously (Versluis et al, 1996).…”

“…At 2, 4 or 8 h after injection, the mice were anaesthetized with diethyl ether, and a 0.4 ml blood sample was taken. The mice were subsequently Britsh Journal of Cancer (1998) 78(12), [1607][1608][1609][1610][1611][1612][1613][1614] (Versluis et al, 1996). Figure 3A and B, and the effects on the degradation of ['12]LDL by the cells are depicted in Figure 3C and D. To examine the role of apoE, both apoE-containing liposomes and liposomes devoid of apoE were used Both LDL and the liposomes were able to inhibit British Jo/rnal of Cancer (1998) LDLr-mediated drug delivery to B 16 tumour by apoE liposomes 1611 Ca' dependent (Goldstein and Brown.…”

“…The B 16 tumour-mouse model was used because the LDL receptor expression on B16 cells has been well characterized (Ponty et al 1993: Versluis et al 1996. We investigated the LDL receptor-mediated interaction of apoE-enriched liposomes with cultured B16 tumour cells.…”