Experimental Study On The Effects Of NGF And Its Receptors On Perineural Invasion Of Bile Duct Carcinoma

Perineural invasion is one of the important features for patients with bile duct carcinoma, and it thus cause the difficulity in curative resection and high recurrence. It was believed that the tumour invasion appeared to follow the plane of least resistance of the perineural space; but recent search shows that some molecules on the surface of cancer cells may have effects on the progression of tumor perineural invasion. It is necessary, therefore, to investigate the molecular mechanisms in patients with perineural invasion. Nerve growth factor(NGF) is a member of the neurotrophic factor(NTF) family. It binds to the cognate receptors(the high-affinity receptor-TrkA and the low-affinity receptor-p75NTR) on the neuron. The NTFs are imperative to the survival, growth and differentiation of important neuronal populations in the peripheral and central nervous systems. But recent research shows that the NTFs and their receptors are implicated in the autocrine and (or) paracrine growth regulation of a number of neuronal and non-neuronal tumor types. For example, the expression of NGF and its receptors is found to be correlated with the perineural invasion of prostate cancer; and another member of NTFs-GDNF induces the pancreatic-cancer cells migrate along the concentration gradient of GDNF produced from peripheral ganglions. Thus, we choose NGF and its receptors under our research to elucidate the mechanisms of perineural invasion in human bile duct carcinoma.Firstly, we investigate the expression of NGF and its receptors- TrkA and p75NTR-in the bile duct carcinoma line QBC939 and 38 cases of resected bile duct carcinoma by immunocytochemical method. We find that there is abundant NGF and TrkA expression in the cell line QBC939, and so does p75NTR. In the 38 specimens of human bile duct carcinoma, The ratio of sections stained by NGF is 61%; the ratio of p75NTR is 50%, and that of TrkA is 71%. The perineural invasion ratio is 55%. The expression of NGF and TrkA has remarkable relation to PNI(perineural invasion index) (P<0.01); and the expression of p75NTR remarkably relate to the differentiation of the tumor cells(P<0.05). It prompts that the expression of NGF and TrkA may play an important role in the perineural invasion of bile duct carcinoma; and the expression of p75NTR may play an important rolein the neoplasm progression of bile duct carcinoma. Thus,we can suppose that the perineural invasion is induced by the autocrinic or paracrinic NGF through the TrkA expessed on the surface of cancer cells. But the mechanism is still unclear.Secondly, the mechanisms of NGF and its receptors on inducing perineural invasion of the QBC939 cell line are investigated:2.1 The results of in vitro cell migration assay show a major effect of chemotaxis and a minor effect of chemokinesis by NGF on the QBC939 cells; the effects are dose-dependent and can be blocked by k-252a, inhibitor of tyrosine receptor kinases which including TrkA. From these results, we can infer that the paracrinic NGF from the perineural tissue may be supplied as the chemotactic factor to induce the directional migration towards neural tissue. And the autocrinic NGF by QBC939 cells might also take part in this procedure. These results can partly explain the neurotropism of bile duct carcinoma.2.2 We use RT-PCR and Western-blotting analysis to evaluate the change of MMP-2 and MMP-9 expression in QBC939 cells induced by 50ng/ml NGF(which is the physical concentration of NGF). The results show that NGF markedly increase the production of MMP-2 and MMP-9 of the QBC939 cells, and this effect can be blocked by k-252a. It is implied that NGF might promote cancer invasion by the enhancement of degradation of the ECM. 2.3 The effect on adhesive capacity to neuron by NGF induced cells.2.3.1 NCAM is conducted to be the "navigating" and "docking" molecule in perineural invasion. Here we use Western-blotting analysis to evaluate the level of NCAM molecule expressed in QBC939 cells induced by 50ng/ml NGF. The results show that NCAM expression are m...