Cell signalling in cancer. A functional and immunochemical investigation of purinergic,adrenergic, nitrergic, and prostaglandin mediated signalling mechanisms in experimental and human cancer

Sammanfattning: Disturbed cell signalling is a hallmark of the cancer cell, being one important mechanismbehind uncontrolled cell growth. In the current study, we have investigated cancer cellsignalling with the focus on some first messenger and/or their receptors, in the murine celllines, MCG 101, and K1735-M2, and the human colon cancer cell line, HT-29. The signallingmolecules investigated were noradrenaline, prostaglandin E2 (PGE2), adenosine-5 L-triphosphate (ATP), and nitric oxide (NO).Functional investigations were undertaken by microphysiometry, which method allows forthe monitoring in real time of extracellular acidification rate (ECAR). This is a measure of theongoing metabolic activity of the cell, and how the challenging of the cell with a ligand mayaffect this variable. In addition, we investigated protein expression by immunochemicalmethods.Noradrenaline caused increased ECAR in the three cell lines. In the MCG 101 and K1735-M2, the membrane receptor involved was found to be the À3-adrenoceptor.PGE2 affected ECAR only in the MCG 101. Moreover, in this cell line, we foundindications of an autocrine loop, in which PGE2 may upregulate cyclooxygenase, the enzymeresponsible for prostanoid synthesis.ATP increased ECAR in the MCG 101, most likely via the P2Y2-purinoceptor subclass.In the HT-29, this ligand caused a biphasic effect on ECAR (increase followed by decrease).Both effects appeared to be elicited via the same receptor; this could be P2Y2, but aninvolvement of also P2Y4 cannot be excluded.In human colon cancer, the P2Y2-, and P2Y4-purinoceptors were significantly overexpressed,compared to adjacent, macroscopically tumour-free colon tissue.The inducible isoform of NO synthase (iNOS) was identified in the MCG 101. The findingthat also nitrotyrosine was expressed in these cells could suggest that iNOS was catalyticallyactive, resulting in the formation of peroxynitrite and, secondarily, nitrotyrosine. The tumourcells were, however, unaffected functionally by the administration of a eNO-donor f (sodiumnitroprusside), which may indicate that NO does not serve a function as e.g. anautocrine/paracrine growth factor for these cells.It is concluded that microphysiometry is a valuable tool for the investigation of functionalreceptors in tumour cells. Of the signalling molecules investigated in the current study, ATPappears to be of considerable interest for e.g. colon cancer.