| Background Pancreatic cancer is one of the most malignant tumors with a rapid progressand an extremely poor prognosis. The rate is stepping up year by year. It is difficult to befound in early stage. At the time of diagnosis, most patients have locally advanced diseaseand distant metastasis. Perineural invasion is a histopathologic characteristic in pancreaticcancer which concerned with many signal molecules secreted by neural tissues and cancercells during the neuro-cancer interactions. The four members of Glial cell line-derivedneurotrophic factor (GDNF) family (GDNF,NTN,ARTN and PSP) and their receptorsare expressed in pancreatic cancer cells. Artemin (ARTN) enhances survival, proliferation,and regeneration of neurons. Additionally, it acts as a guidance molecule that inducesmigration and axonal projection from neurons. Artemin is abnormally expressed inpancreatic cancer, breast cancer, non-small cell lung cancer and endometrial cancer and haseffects on the oncogenicity and invasiveness of these cancer cells.Objective To investigate the biologic effects of Artemin on the pancreatic cancer growthand invasion in cell line, human tissues and animal models. To approach the potentialtherapeutic effects on pancreatic carcinoma by targeted retrain Artemin protein level.Methods1. Artemin protein level was detected in11samples of pancreatic carcinoma tissues, inthe respective normal tissues adjacent to the cancer and in6kinds of pancreatic carcinomacell lines by Western blot analysis. Immunohistochemisty (IHC) was applied on tissuearray slides of100cases of pancreatic carcinoma (60cases of pancreatic ductaladenocarcinoma and40cases of adenosquamous carcinoma) and23cases of normal tissuenear the cancer to screen the distribution of Artemin. The relationship was also analyzedbetween the positive expression of Artemin and perineural invasion, metastasis, tumordifferentiation and other indicators of clinical pathology of pancreatic carcinoma. Growth associated protein43(GAP43)was stained by IHC in the tissue array slides to quantifynerve density and area in stroma. The relationship was investigated between Arteminexpression and hypertrophy and hyperplasia of stromal nerve fibers.2. An Artemin eukaryotic expression plasmid and4kinds of microRNA plasmids wereconstructed and transfected into Panc1cell line with liposome system to establish thestable expression monoclonal cell lines. The cell lines which expressed the highest level ofArtemin and the lowest level of Artemin were screening out by Western blot analysis andqRT-PCR analysis. MTT-growth curve, flowcytometry analysis, wound healing assay andtranswell chamber Matrigel-invasion assays were used to detect the effect of Artemin oncancer cell proliferation, cell cycle, apoptosis and invasion of Panc1. To evaluate whetherpancreatic cell line features neurotrophic attributes by secret Artemin protein, thesupernatants from Artemin over expressed cell line and Artemin RNA interfered cell linewere collected to treat PC12cell line respectively. The cell growth and neuronaldifferentiation of different groups were observed and compared.3. Orthotopic animal models for pancreatic cancer were set up with the Arteminoverexpressed cell line and Artemin RNA interfere cell line building in part2. The grossand microscopic characters were observed and compared. GAP43was also stained by IHCto compare the hyperplastic situations of stromal nerve fibers.Results1. Artemin was detected at enhanced levels in pancreatic carcinoma compared withnormal pancreas near the cancer. It was also detected in6kinds of pancreatic cancer celllines at different levels. Immunohistochemical detection showed that the positiveexpression rate in carcinoma (71%) was significantly higher than that in normal tissuesadjacent to cancer (43.5%). The positive expression localized predominantly in cancer cellsand tubular complex adjacent to cancer, as well as in hypertrophic nerves and arterial walls.The positive expression of Artemin was significantly related to the lymphatic metastasisand perineural invasion, but was not related to pathological type,sex, age, tumor location,tumor size, tumor differentiation, and surrounding organs invasion. Neuropathic alterationswere obvious in carcinoma stroma, including hypertrophy and hyperplasia of neuron fibers.More dense neural networks and enlarged nerves were detected in Artemin positiveexpressed cases (density:49.72±5.177/cm2; area:0.328±0.058mm2) than those in Arteminnegative expressed cases (density:28.72±4.248/cm2; area:0.0857±0.025mm2).2. Artemin over expressed cell line had increased cancer cell colony formation than control vector group, while Artemin RNAi decreased colony formation than control.Wound healing assay showed increased cell migration in Artemin over expressed cell linethan that in control. Matrigel-invasion assays showed that Artemin promoted cancer cellinvasion and Artemin RNAi can inhibit the effect. MTT-growth curve, cell cycle andapoptosis had no difference between different groups. The Artemin over expressed cellculture supernatants can induce PC12cell neuron-like differentiation.3. Nude mice implanted with Artemin over expressed cells had increased size of thetumor compared with the vector control group. Artemin over expression can promotecancer cell invasion into surrounding organs as well as increase perineural invasion rateand vascular invasion rate, and promote the adjacent nerve fiber hyperplasia. The ArteminRNAi group had decreased tumor formation rate and smaller tumor volume than control.Surrounding organs invasion was decreased. Vascular invasion, lymph node metastasis andperineural invasion were not found.Conclusion1. The expression of Artemin in pancreatic carcinoma involves in neuralpathological changes in cancer stroma, such as neural fibers hypertrophy and hyperplasia,and it also related to lymph node metastasis and perineural invasion.2. The expression ofArtemin in pancreatic cancer cell line can promote cell survival, enhance cell migrationand invasion. And it features the cancer cells neurotrophic attributes through paracrinemechanisms. Artemin RNA interference can significantly inhibit these effects.3. Theexpression of Artemin in pancreatic cancer cell can increase the volume of orthotopictransplantation tumor in vitro, promote cancer cells invasion into surrounding organs,vasculars and nerves, and stimulate stroma nerve fiber hyperplasia. Targeted inhibition ofArtemin by microRNA has obvious inhibitory effect on pancreatic cancer growth andinvasion. Artemin may become a new potential target for pancreatic cancer treatment.
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