Hepatocyte growth factor (HGF), heat shock proteins (HSPs) and multidrug resistance protein (MRP) expression in co-culture of colon tumor spheroids with normal cells after incubation with interleukin-1β (IL-1 β) and/or camptothecin (CPT-11).

Tumor chemoresistance and metastasis are some of the most important problems in colon cancer therapy. In the present study, co-cultures of human colon carcinoma cell spheroids, obtained from different grades of tumor, with human colon epithelium, myofibroblast and endothelial cell monolayers were performed. The purpose of these co-cultures was to reflect, in in vitro conditions, different stages of colon tumor development. In order to investigate the invasive capacities of the tumor cells and their resistance to chemotherapy, HGF, HSP27, HSP72 and MRP levels were analyzed after incubation of the co-cultures with IL-1 and irinotecan (CPT-11) added as single agents or in combination. Myofibroblasts produced significantly higher amounts of HGF than epithelial cells. Tumor cells released trace amounts of this molecule. In co-cultures, IL-1 induced HGF release, while CPT-11 alone or combined with IL-1decreased HGF secretion. An immunoblotting analysis followed by densitometry revealed that the combination of IL-1 plus CPT-11 added to the co-cultures led to a decrease in HSPs and MRP levels. In conclusion, direct and paracrine interactions of colon tumor cell spheroids with normal cells and exogenously added CPT-11 change HSP27, HSP72 and MRP expression in comparison to monocultures. IL-1 and CPT-11, dependent on whether they are added separately or jointly, differentially modulate HGF expression in monocultures of colon tumor spheroids or normal cells and their co-cultures.

Expression of HSP27, HSP72 and MRP proteins in in vitro co-culture of colon tumour cell spheroids with normal cells after incubation with rhTGF-ß1 and/or CPT-11.

We studied the expression of inducible heat shock protein (HSP27, HSP72) and multidrug-resistance protein (MRP) in co-cultures of human colon carcinoma cell spheroids obtained from different grades of tumour with normal human colon epithelium, myofi broblast and endothelial cell monolayers. We also measured the infl uence of recombinant human transforming growth factor β1 (rhTGF-β1) and camptothecin (CPT-11), added as single agents or in combination, on the levels of the HSPs, MRP, interleukin (IL)-6 and nitric oxide (NO). An immunoblotting analysis with densitometry showed that rhTGF-β1 and/or CPT-11 increased HSP27, HSP72 and MRP expression in tumour cells and myofi broblasts, as well as in co-cultures compared with appropriate controls. By contrast, in colonic epithelium, inhibition of HSPs and MRP was comparable with that of the control. In endothelial cells, HSP72 was undetectable. Direct interaction of colon tumour spheroids with normal myofi broblasts caused a signifi cant, tumour-grade dependent increase in IL-6 production. Production of IL-6 was signifi cantly lowered by rhTGF-β1 and/or CPT-11. Tumour cell spheroids cultivated alone produced larger amounts of NO than normal cells. In co-culture, the level of the radical decreased compared with the sum of NO produced by the monocultures of the two types of cells. rhTGF-β1 and/or CPT-11 decreased NO production both in tumour and normal cell monocultures and their co-cultures. In conclusion, direct interactions between tumour and normal cells infl uence the expression of HSP27, HSP72 and MRP, and alter IL-6 and NO production. rhTGF-β1 and/or CPT-11 may potentate resistance to chemotherapy by increasing HSP and MRP expression but, on the other hand, they may limit tumour cell spread by decreasing the level of some soluble mediators of infl ammation (IL-6 and NO).