Role of lipid-mobilising factor (LMF) in protecting tumour cells from oxidative damage

Abstract

Lipid-mobilising factor (LMF) is produced by cachexia-inducing tumours and is involved in the degradation of adipose tissue, with increased oxidation of the released fatty acids through an induction of uncoupling protein (UCP) expression. Since UCP-2 is thought to be involved in the detoxification of free radicals if LMF induced UCP-2 expression in tumour cells, it might attenuate free radical toxicity. As a model system we have used MAC13 tumour cells, which do not produce LMF. Addition of LMF caused a concentration-dependent increase in UCP-2 expression, as determined by immunoblotting. This effect was attenuated by the beta3 antagonist SR59230A, suggesting that it was mediated through a beta3 adrenoreceptor. Co-incubation of LMF with MAC13 cells reduced the growth-inhibitory effects of bleomycin, paraquat and hydrogen peroxide, known to be free radical generators, but not chlorambucil, an alkylating agent. There was no effect of LMF alone on cellular proliferation. These results indicate that LMF antagonises the antiproliferative effect of agents working through a free radical mechanism, and may partly explain the unresponsiveness to the chemotherapy of cachexia-inducing tumours.

Figure 1

SDS–PAGE of LMF purified from human urine according to the protocol described in Materials and methods. Lane 1, MW markers; lane 2, LMF. Detection was by Coomassie brilliant blue stain.

Figure 2

Immunoblot of hZAG (lanes 1–3) and hLMF (lanes 4–6) (10 μg) detected with polyclonal antibody to hZAG.

Figure 3

Immunoblot of soluble extracts of MAC13 (lanes 1–3) and MAC16 (lanes 4–6) detected with monoclonal antibody to human ZAG.

Figure 4

Immunoblot of UCP-2 expression in MAC13 cell line (A) in the presence of 0 (lane 1), 0.23 (lane 2), 0.35 (lane 3) and 0.58 (lane 4) μM LMF after 24 h incubation and (B) in the presence of 0 (lanes 1 and 6), 0.23 (lanes 2 and 7), 0.35 (lanes 3 and 8), 0.46 (lanes 4 and 9) and 0.58 (lanes 5 and 10) μM LMF for 24 h in the absence (lanes 1–5) or presence (lanes 6–10) of 10 μM SR59230A. (C) Densitometric analysis of the blot shown in (B). The symbols ♦ are in the absence of SR59230A and ▪ in the presence; n=3. Differences from values in the presence of SR59230A are indicated as b, P<0.01.

Figure 5

Effect of bleomycin alone on the growth of MAC13 cells (open boxes), or in the presence of 0.58 μM LMF (hatched boxes), or 0.58 μM LMF+10 μM SR59230A (stippled boxes). Zinc-α2-glycoprotein alone was used at a concentration of 0.58 μM. Total repeats n=3. Differences from values in the presence of bleomycin alone are indicated as c, P<0.001, white differences from bleomycin +ZAG are indicated as b, P<0.01, and d, P<0.001.

Figure 6

Effect of hydrogen peroxide on the growth of MAC13 cells alone (open boxes) and in the presence of 0.58 μM ZAG (hatched boxes). Zinc-α2-glycoprotein alone was used at a concentration of 0.58 μM. Total repeats n=3. Differences from values in the presence of hydrogen peroxide alone are indicated as a, P<0.05, and b, P<0.01.

Figure 7

(A) Effect of paraquat on the growth of MAC13 cells alone (open boxes) and in the presence of 0.58 μM LMF alone (hatched boxes) or with 10 μM SR59230A (stippled boxes). Lipid-mobilising factor was used alone at a concentration of 0.58 μM. Total repeats n=3. Differences from values in the presence of paraquat alone are indicated as a, P<0.05, and b, P<0.01, white differences from paraquat+LMF are indicated as c, P<0.001. (B) Levels of MDA in MAC13 cells after no treatment (C) or treatment with 0.1 μM paraquat (P), 0.1 μM paraquat+0.58 μM ZAG (P+ZAG) or 0.1 μM paraquat+0.58 μM ZAG+10 μM SR59230A (P+ZAG+SR); n=6. Differences from control are shown as a, P<0.05, while differences from 0.1 μM paraquat are shown as b, P<0.001, and differences from P+ZAG are shown as c, P<0.001.

Figure 8

Effect of chlorambucil on the growth of MAC13 cells alone (open boxes) and in the presence of 0.58 μM LMF (hatched boxes). Lipid-mobilising factor was used alone at a concentration of 0.58 μM. There were no significant differences between the two groups.