Effects And Mechanisms Of Esophageal Nerves And Neurotransmitters On Esophageal Cancer Cell Proliferation And Differentiation

1 BACKGROUNDLinzhou City (formerly Linxian County), together with nearby counties in Henan,has been well recognized as the highest morbidity and mortality area for esophagealcancer (EC) in the world. Moreover, EC remains currently the leading cause ofcancer-related death in these areas. Nevertheless, the molecular mechanism ofesophageal carcinogenesis is largely unknown. Recent studies have indicated that thegeneration and development of EC are correlated with the individual's psychologicalstress and neuroimmunologic factors.The viscera effector cells can be regulated by the nervous system viacenter-peripheral autonomic nervous (sympathetic nerve and vagus nerve) pathway. Ithas been reported that there is hypogenesis of vagus nerve and recurrent laryngealnerve in esophageal atresia patients. And animal experiments also demonstrated thatthe removal of either cervical nervous crest or cervical sympathetic ganglia can leadto esophageal mucosa absence and esophageal atresia, which indicates thatesophageal diseases are closely related to esophageal nerves both in structure and function. Recent clinical researches indicate that EC exsection with vagus nervereserved could accelerate postoperative recovery and procrastinate the tumor relapse.Western scholars have carded out a great deal of researches and presume thatgastroesophageal reflux disease (GERD) resulting from functional disorder ofesophageal motor may contribute to esophageal carcinogenesis, which indicates thatthe functional disorder or defection of vagus nerve may play an important role inesophageal carcinogenesis.The acetylcholine (ACh) and norepinephrine (NE) released fromparasympathetic fibers of vagus nerve and sympathetic fibers and their receptors carryout the regulation of esophageal function. Neurobiological researches demonstrate thatacetylcholine receptors (AChRs) have significant physiological functions on cell proliferation,differentiation and formation of cytoskeleton. Nicotine, the excitomotor of AChRs, prompts theproliferation of gliacyte tumor while tubocurarine (TC) the antagon inhibits that. Clinicalinvestigations have shown that the generations of malignant tumors were correlatedwith dysfunction of mAChR. And it is also reported that NE can regulate the differential typeof central neuron and induce the apoptosis of alveolar cell. But there is no report about the effectof nerves and corresponding transmitters on esophageal carcinogenesis. We presume thatesophageal carcinogenesis is a multistage progressive process involved by multiple factors,multiple genetic changes and their complicated mechanism. Researches of years in ourlaboratory also indicate that a great deal of anti-oncogenes and oncogenes and the functionaldisorders of proteins are the original causes of esophageal carcinoma, but the key genes andprotein molecules haven't been identified. It is discovered from recent census andfollow-up visits that the predilection sites for esophageal carcinogenesis are the middleand inferior segments, and the character of precancerous lesions are of two-waydevelopment, which indicates that the carcinogenesis is reversible. Other studies which find thatthe activation of the brainstem 5-HT (5-hydroxytryptamine, 5-HT) rapheal nuclei-vagus pathwaycan protect the viscera manifested that the rapheal nuclei 5-HT neuron regulates the vagus nerveby releasing 5-HT. Researches on psychological stress discovered that long-standing mental stressor depression leads to descending of center 5-HT and its receptor level, indicating that stress maydegrade the cholinergic function from vagus nerve to esophagus, thus it can be inferred that the esophageal pathological changes may concern the structural and functional abnormality of theneural pathway from vagus nerve to esophagus, and that the disfunction of the esophageal nervetakes a significant place in esophageal cancerization and the malignant transformation ofesophageal carcinoma cells. Recent studies have indicated that EC cell has the feature ofabnormally expressing Keratins, Peroxiredoxin-1 (PRX1) and calcium-dependent lipid bindingprotein-2 (Annexin-2). Keratin 5 (KS) and Keratin 20 (K20) are the marks of progenitorepithelium cells and mature cells respectively, and they are also the foundation of epithelium celladhering, sustaining and protecting functions. PRX1 has repairing function to damaged DNA;Annexin-2 can suppress cells' proliferation and regulate cells' differentiation and it may be one ofthe important molecules inhibiting esophageal carcinogenesis. AChR and Cholineacetyltransferase (CHAT) are transmitting molecules by which ACh acts on cells.Microtubule-associated protein-2 (MAP2) is a kind of skeleton connection protein in the neuron,the expression of which may be decreased in degenerating neurons and nerve fibers.These molecules mentioned here are provided as observational indexes for furtherresearch.Therefore, the design of this research adopts the methods and techniques of tissues sliced, HEstraining, immunofluorescence (IMF), immunohistochemical straining and so on to survey thedistribution features of esophageal nerves, the intramural neuroplexus morphous of the esophagealcarcinoma, and the changes of the functional genes and protein expression, and to analyze therelationship between those indexes mentioned above and the carcinogenesis and celltransformation.Furthermore, we use vitro cell culture, immunofluorescence, immunohistochemicalstraining, flow cytometry, RT-PCR etc. to make an exploratory study on the affection of NE, thesympathetic transmitter, and ACh, the parasympathetic transmitter, on the changes inanti-oncogenes and functional protein expression of the esophageal carcinoma, thus we cananalyze the effects of NE and ACh on esophageal carcinogenesis and cell transformation, which isof significance in revealing and illuminating the molecular mechanism of esophagealcarcinogenesis, and providing new research ideas on target for cancer prevention and treatment.2 MATERIAL and METHODS2.1 Observation of the relationship between EC intramural nerve morphous and expression changes of cancer cellular functional proteins.There were 42 EC specimens that were all from Central Hospital in Linzhou orYaocun EC Hospital and none of the patients had accepted any chemotherapy orradiotherapy. The 42 patients included 37 males and 5 females, whose mean age was60.3±6.5. After 4% polyformalin fixation, paraffin imbedding, serial sectioning anddying the cancer tissue, para (adjacent) carcinoma tissue and normal tissue via IMFand immunohistochemical ABC method, we surveyed and analyzed the relationsamong intramural nerve morphous of the three kinds of tissues and AChR, CHAT,Annexin-2, PRX1, MAP2, Keratin 5 and Keratin 20 expression changes of the cancercells, including relations among expression changes of every functional proteins, so asto identify the change pattern and the effect of esophageal nerves in the process ofesophageal carcinogenesis.2.2 Experiment of cell culture in vitroBy adding cell receptor blocking agents Atropine and Tolazoline and xtracellularmatrix (ECM) blocking agent U0126, we cultured in vitro the human esophagealcarcinoma cell strain EC9706, the ESCC (esophageal squamous carcinoma cell,ESCC) strain preserved in the Cancer Lab of the Medical College of ZhengzhouUniversity. By means of immunohistochemical straining, IMF, flat cell clone, flowcytometry, fluorescently quantitative RT-PCR and so on, we observed the changes ofthe cell cycle and vigor of cell generation as well as the changes of the expression ofAChR, CHAT, Annexin-2, Keratin 5 and Keratin 20 in EC9706 both before and afterthe treatment of ACh and NE, then analyzed the affection of ACh, NE and ECM to theproliferation and differentiation of EC cells and their mutual relationship, so as todetect the effect of neurotransmittcr in the process of esophageal carcinogenesis andcancer cell transformation and find the possible molecular mechanism.2.3 Statistics analysisThe data is analyzed with SPSS10.0 by means of one-factor analysis of varianceand paired sample t-test. It is described as (?)±s, with a=0.05 as the size of test andP＜0.05 meaning the variance to be significant. 3 RESULTS3.1 The relation between esophageal intramural nerve morphous and expressionchanges of cancer cellular functional proteinsThe MAP2 positive intensity of esophageal intramural nerve decreases graduallyin the order of normal tissue, hyperplasia, preinvasive carcinoma and cancer, whilethe fine nerve fibers reduce and finally disappear. The expressions of AChR, CHAT,Annexin-2, PRX1, and Keratin 20 decrease gradually along with the development ofcarcinogenesis and the aggravation of esophageal intramural nerve retrogradation,while Keratin5 changes less in the process of carcinogenesis. The change ofKeratin20,Annexin-2 expression keeps the same with PRX1. The expression ofAChR is typically to decrease gradually from cell membrane to cytoplasm and thenshift or concentrate from cytoplasm into nuclear with the development ofcarcinogenesis. ChAT changes and decreases late in the process of carcinogenesis" andmalignant transformation and expresses excessively in the atypical hyperplasia phase.3.2 The effect of ACh and NE on the cell proliferation and differentiation ofEC9706, the human esophageal carcinoma cell line cultured in vitro3.2.1 The effect of ACh and NE on the cell proliferation of EC9706, theesophageal carcinoma cell line in vitroAfter treatment of ACh and NE, the cell soma was augmented, the processeserupted and the nuclear-cytoplasmic ratio was decrease. The esophageal carcinomacells grow with lower density and have longer processes under the role of NE in vitro.The process length of ACh and NE groups have highly significant differencecompared with the other groups (P＜0.01). With mAChR and a-receptor blocked byAtropine and Tolazoline respectively, ACh and NE remain to promote the growth ofcell processes, and the two groups have significant difference compared with thecontrol groups (P＜0.05). The nuclear-cytoplasmic ratio of ACh and NE groups havesignificant difference compared with the control groups (P＜0.05). With receptorsblocked in ACh and NE groups, the nuclear-cytoplasmic ratio increases a little but does not have significant difference (P＞0.05).3.2.2 The effect of ACh and NE on the cell proliferation activity of EC9706, theesophageal carcinoma cell line in vitroBoth ACh and NE can degrade the cell proliferation activity of EC in vitro, andhave significant difference compared with the control groups (P＜0.05), and can stilldegrade the cell proliferation activity of EC in vitro with mAChR and a-receptorblocked.3.2.3 The effect of ACh and NE on the cdi proliferation inhibition ratio ofEC9706, the esophageal carcinoma cdl line in vitroACh and NE can elevate the cell proliferation inhibition ratio of EC9706 theesophageal carcinoma cell line in vitro, and have the effect of restraining the cellproliferation. The groups have significant difference compared with the controlgroups (P＜0.05), and the inhibition ratio of the colony formation descends withreceptors blocked Atropine and Tolazoline.3.2.4 The effect of ACh and NE on the AChR, CHAT, PRX1, Annexin-2, Keratin5and K20 expression of EC9706 the esophageal carcinoma cell line in vitroACh and NE can obviously promote the AChR, CHAT, PRX1, Annexin-2,Keratin5 and K20 expression of EC9706 the esophageal carcinoma cell line in vitro,and the two groups have significant difference compared with the control groups (P＜0.05). And they can still promote the functional proteins expression of theesophageal carcinoma cell in vitro with mAChR and a-receptor blocked; moreoverwith mAChR blocked by atropine, the ACh groups still have significant differencecompared with the control groups (P＜0.05).3.2.5 The effect of ACh and NE on the cell cycle and apoptosis of EC9706 theesophageal carcinoma cell line in vitroACh and NE can block the cell cycle, making the cells stopped in G1 phase andG2 phase, and reduce the carcinoma cell population in S phase compared with thecontrol groups. The blockage effect is in a descending order: ACh+NE＞NE+Tolazoline＞NE＞ACh+Atropine＞ACh, and the blockage ratio is respectively 90％, 70％, 15％, 15％, 10％compared with the control groups. ACh and NE caninduce the apoptosis of the esophageal carcinoma cell in vitro, and the apoptosis ratioof the carcinoma cells induced is respectively 30 times(ACh group), 4 times (ACh+Atropine group), 10 times (NE group), 20 times (NE+Tolazoline group), and 30times (ACh+NE group) that of the control group.3.2.6 The effect of ACh and NE on the Annexin-2 mRNA expression of EC9706the esophageal carcinoma cell line in vitroACh and NE can promote the multiplication of Annexin-2 mRNA expression inEC9706 the esophageal carcinoma cell line in vitro, and the two groups havesignificant difference compared with the control groups (P＜0.05). The Annexin-2mRNA expression level of ACh group, ACh+Atropine group, ACh+NE group, NEgroup and NE+Tolazoline group is respectively 20 times, 8 times (NE group), 3 times,2 times, and 4 times higher than that of the control group.3.3 The effect of ACh and NE on the proliferation and differentiation of EC9706cells with ERK1/2 blockedAs ERK1/2 blocked, there arc some changes with the morphous of EC9706 theesophageal carcinoma cell line in vitro, the processes becoming short andnuclear-cytoplasmic ratio augmenting, and that group has significant differencecompared with the control groups (P＜0.05). With ERK1/2 blocked, ACh and NEdecrease the nuclear-cytoplasmic ratio, which is significant statistically (P＜0.05),and the short processes grow a little longer, which is not significant statistically (P＞0.05). With ERK1/2 blocked, the expression of AChR, CHAT, Anncxin-2 and Keratin20 in EC9706 the esophageal carcinoma cell strain in vitro decreases significantly (P＜0.05), while in the groups added ACh and NE, the descended expression increasesrelatively and that is significant statistically (P＜0.05). With ERK1/2 blocked, the cellproliferation activity in EC9706 the esophageal carcinoma cell line in vitro decreasessignificantly (P＜0.05) and the cell proliferation inhibition ratio increasessignificantly, while in the groups added ACh and NE, the effect is further enhanced.With ERK1/2 blocked in EC9706 the esophageal carcinoma cell line in vitro, the cellsare stopped in S phase with the population increasing and percentage of apoptosis cells decreased, while ACh and NE can further reduce the cell percentage resultingfrom ERK1/2 blocked in S phrase (period of cell cycle). The intensity of blockageeffect ranks in a descending order: ACh+NE＞ACh+A＞ACh＞NE+Tolazoline＞NE.4 CONCLUSIONS4.1 During the process of carcinogenesis development, the esophageal intramuralnerves decreased gradually or disappeared and the expression of AChR, CHAT,Annexin-2, PRX1 and K20 reduced gradually as well. These findings indicate that thedecrease or dysfunction of intramural nerves may be the nervous factors of theesophageal carcinogenesis. With the generation and development of carcinogenesis,the change pattern of AChR is that the expression decreases gradually in cellmembrane and cytoplasm at first, and then shifts or concentrates from cytoplasm intonuclear, which is possibly the early receptor event that reduces sensitivity of epithelialcells in esophageal mucosa to nervous ACh and furthermore leads to the priming ofcarcinogenesis. The elevation of PRX1 might mean the active molecules that protectthe cells stimulated by injuries keep Annexin-2 and Keratin20 to express, while thedecrease of PRX1 might be the internal cause that leads to the descent ofself-protecting and self-stabilizing function and promotes the carcinogenesis andmalignant transformation.4.2 ACh and NE could induce cell differentiation of EC9706 (the esophagealcarcinoma cell line) in vitro, and still could promote these cells differentiation afterthe mAChR and a receptor were blocked with Atropine and Tolazoline respectively.These findings demonstrate that the esophageal carcinoma cell could be induced orincreased its ability to differentiate, making the soma augment, the processes eruptand the nuclear-cytoplasmic ratio descend, and the expression of functional proteinsincrease. The mechanism may be through nAChR andβreceptor.4.3 Both ACh and NE can degrade the cell proliferation activity of EC in vitro. Itappears that they induce carcinoma cell converted from S phase to G1 phase and G2phase, or block the cycle and affect the cell proliferation. This effect is regulated bymulti-receptor and multi-path. 4.4 ACh and NE can promote the multiplication of Annexin-2 mRNA expression inEC9706 the esophageal carcinoma cell line in vitro. ACh has the most powerful effectand augments the expression by 20 times, and ACh +Atropine 8 times, NE,NE+Tolazoline and ACh+NE has feeble effect and can only increase the expressionby 2～4 times.4.5 ERK1/2 blocker degrades the cell proliferation vigor as well as restrains the celldifferentiation. ACh and NE enhance the degradation of the cell proliferation vigor ofEC9706 the esophageal carcinoma cell line in vitro, with ERK1/2 blocked, and theyinduce the cell differentiation by regulating the functional proteins expression ofcarcinoma cell. The discovery of the mutual relationship of the neurotransmitter andthe ECM blocker will provide an original research idea and direction for furtherrevealing the molecular mechanism of EC generation and development in vivo, andprovide new targets for the drug design used on prevention and cure of the esophagealcarcinoma.