Leptin stimulates the proliferation of human colon cancer cells in vitro but does not promote the growth of colon cancer xenografts in nude mice or intestinal tumorigenesis in Apc(Min/+) mice - PubMed (original) (raw)
Leptin stimulates the proliferation of human colon cancer cells in vitro but does not promote the growth of colon cancer xenografts in nude mice or intestinal tumorigenesis in Apc(Min/+) mice
T Aparicio et al. Gut. 2005 Aug.
Abstract
Background and aims: Leptin, the product of the ob gene, has been suggested to increase the risk of colon cancer. However, we have shown that although leptin stimulates epithelial cell proliferation it reduces the development of carcinogen induced preneoplastic lesions in the rat colon. Here, we explored the effect of leptin in vitro on proliferation of human colon cancer cells, and in vivo on the growth of HT-29 xenografts in nude mice and the development of intestinal tumours in Apc(Min/+) mice.
Methods: Proliferation of HT-29, LoVo, Caco2, and SW 480 cells was assessed in the absence or presence of leptin (20-500 ng/ml) by 3H-thymidine incorporation and cell count. Leptin (800 microg/kg/day) or its vehicle was delivered for four weeks to nude mice, inoculated with HT-29 cells on day 0, and for six weeks to Apc(Min/+) mice.
Results: Leptin dose dependently stimulated cell DNA synthesis and growth in all cell lines. In nude mice, leptin caused a 4.3-fold increase in plasma leptin levels compared with pair fed controls. This hyperleptinaemia, despite leptin receptor expression in tumours, did not induce significant variation in tumour volume or weight. Tumour Ki-67 index was even inhibited. In leptin treated Apc(Min/+) mice, a 2.4-fold increase in plasma leptin levels did not modify the number, size, or distribution of intestinal adenomas compared with pair fed controls.
Conclusions: Leptin acts as a growth factor on colon cancer cells in vitro but does not promote tumour growth in vivo in the two models tested. These findings do not support a pivotal role for hyperleptinaemia in intestinal carcinogenesis.
Figures
Figure 1
Biological effects of leptin in the human colonic HT-29, LoVo, Caco-2, and SW 480 cell lines. (A) Tyrosine phosphorylation assay of Ob-R leptin receptor in HT-29 cells in the absence or presence of 100 ng/ml of leptin. Ob-Rb was immunoprecipitated with the M18 anti-Ob-R antibody and its phosphorylation was detected by western blot with the 4G10 antiphosphotyrosine antibody (P-tyr). The functional isoform Ob-Rb was identified as a protein band of approximately 120 kDa. Its phosphorylation was maximal after two minutes of incubation with leptin. (B) DNA synthesis and cell growth of HT-29, LoVo, Caco-2, and SW 480 human colonic cancer cells cultured in serum free medium in the absence (control) or presence of 20–500 ng/ml leptin. Each histogram is representative of 2–3 experiments, except for HT-29 cells which served as controls and were studied only once. *p<0.05 ; **p<0.03 to p<0.02; ***p<0.005 to p<0.002 versus controls.
Figure 2
Effect of four week continuous subcutaneous infusion of leptin on the growth of HT-29 cells xenografted in nude mice. (A) Tumour volume during the course of treatment. (B) Tumour weight on day 28, at the time of sacrifice. (C) Proliferative index. (D) Apoptotic index in epithelial tumour cells at day 28. *p = 0.02, **p<0.008 versus vehicle treated nude mice.
Figure 3
Effect of leptin treatment on expression of Ob-Rb and on the mitotic activity of HT-29 tumour xenografts. (A) Western immunoblots of Ob-Rb from homogenates of HT-29 tumour xenografts in leptin and vehicle treated nude mice using M18 Ob-R antibody. A band of approximately 120 kDa corresponding to the Ob-Rb functional isoform of the leptin receptor was identified in all tumours. This band disappeared after immunoneutralisation of Ob-R antibody with the corresponding blocking peptide (not shown). Membranes were reprobed with an antibody against α-tubulin (diluted 1:1000) to assess the relative equal loading of protein samples. (B) Ob-R immunohistochemistry. Insets illustrate detail of labelling on membranes and in the cytoplasm of tumour epithelial cells. (C) Ki-67 immunohistochemistry in tumours (MIB antibody) showing a higher proliferative index in the tumour of a vehicle treated mouse than in that of a leptin treated mouse. Bar = 20 µm.
Figure 4
Effects of subcutaneous leptin treatment on leptinaemia and insulinaemia in nude mice and ApcMin/+ mice. (A) Plasma leptin and (B) plasma insulin after 28 days of subcutaneous continuous infusion of leptin or its vehicle in nude mice. *p = 0.008, **p<0.0003 versus vehicle treated mice. (C) Plasma leptin and (D) plasma insulin after 42 days of subcutaneous continuous infusion of leptin or its vehicle in ApcMin/+ mice. These variables were also measured in C57BL/6J wild mice studied as controls for Apc mutation. *p_<_0.03 versus vehicle treated mice (U test).
Figure 5
Characteristics and Ob-R expression in intestinal adenomas in ApcMin/+ mice. Macroscopic aspect of polyps, singly (A) or in clusters (B). (C) Histological pattern of adenoma (here a sessile duodenal polyp). The duodenal mucosa under the polyp appears atrophic (arrowhead). (D) Detail of the polyp showing areas of moderate to high dysplasia. Asterisks indicate glandular tubes only slightly modified. (E–H) Immunohistochemical Ob-R signal. (E) Part of the duodenal polyp. Labelling is seen in the epithelium surrounding the adenoma (arrowhead) and in the duodenal villi (arrow). (F) Colonic adenoma. Labelling is seen in the surrounding epithelium (arrowhead) and in some dysplastic glandular tubes (arrows). (G–H) Details of the reaction in the duodenal polyp showing heterogeneous Ob-R expression in the surrounding epithelium (G) and in epithelial cells of dysplastic glands in another area (H). Bar = 40 µm.
Figure 6
Numbers and size distribution of adenomas in the small intestine of leptin and vehicle treated ApcMin/+ mice. (A) No difference was observed in the total number or number per cm of intestine (here considering the length before fixation) between the two groups. By averaging values in all females and all males, the total number of adenomas was found to be significantly higher in the small intestine of females than in males (p<0.03). (B) Histogram showing the distribution profile of adenomas in the small intestine as a function of their size. Polyps were classified into six classes. There was no difference between leptin and vehicle treated ApcMin/+ mice for any class. In this study, the size of most polyps ranged between 500 µm and 1.5 mm.
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