The Study Of Acetylcholine Autocrine System In Hepatocellular Carcinoma And Lung Cancer

Hepatocellular carcinoma (HCC) and lung cancer (LC) are highly prevalent and lethal malignancies. Up to now, the prognosis of these cancer patients is far from satisfactory. The major reasons are the poor understanding of the molecular mechanism about carcinogenesis and progression of these cancers, which would possibly provide the rational basis for design of more effective drugs or relevant therapeutic measures. In our laboratory, Wan et al carried out a large scale cDNA transfection screening based on stimulatory or inhibitory effect on cancer and NIH/3T3 cells. After transfection assay of about 30,000 cDNA clones, 38 genes were identified to have stimulatory or inhibitory effect on cell growth, among which the nicotinyl acetylcholine receptor (nAChR) was one of these growth-related genes. Additionally, the alterations of some neurotransmitter-related components have been reported to be closely associated with cancer formation and/or progression. In the present study, we described the results about the autocrine system of the non-neuronal neurotransmitter acetylcholine (ACh) in HCC and LC cells, including their synthesis, degradation, transport, receptor and signal transduction reltated to cancer cell growth and apoptosis.1. Autocrine ACh system in HCC and NSCLCACh autocrine system is a well-known stimulatory neural pathway in neurons. The main components of ACh system include Ach, its receptors (nicotinic receptors nAChR and muscrutic receptors mAChRs), choline acetyltransferase (CHAT), vesicle ACh transporter (VAChT) and acetylcholine degradatation enzyme (acetylcholinesterase, AChE), etc.Fist of all, we examined the expression status of nAChR, mAChR, CHAT and AChE in HCC cell lines, HCC tissues as well as noncancerous liver and normal liver tissues by RT-PCR and Western blot. Results indicated all these molecules were expressed in HCC cell lines and tissues mentioned above. By using cDNA array and real-time-PCR to analyze 15 paried of HCC and matched noncancerous liver tissues, AChE was found as the only molecule which was differentially down-regulated in HCC versus noncancerous liver counterpart. In 5 normal liver tissues, the AChE expression lever was much higher than HCC and noncancerous liver tissues.The down-regulation of AChE expression was further confirmed at protein level by the results from tissue array and immunohistochemistry analysis. These findings made us to conclude that HCC cells behaved like cholinergic phenotype and high level of ACh was present in HCC cancer cells and possibly also in their in vivo microenviroment.The next step is to confirm the status of ACh in HCC and LC cells by using different techniques. By using HPLC-MS/MS analysis of HCC and LC cell lines, ACh can be detected. Though no difference was found in ACh quantity between HCC and noncancerous liver tissues based on the data from limited number of samples (7 pairs), the ACh is definitely present in human HCC tissues. More notably, we analyze the culture medium of HCC and LC cells by highly sensitive HPLC-ECD. We did find ACh is difinitely present in cultured medium; while no ACh is found in cell-free medium.The addition of specific AChE inhibitor, neostigmine (Neo) can remarkably enhance the level of ACh (P<0.05). These data indicate the secretion of ACh from cancer cells.Further more, we also examined the status of ACh autocrine system in other non-neuroendocrine LC and breast cancer. CHAT, VAChT and al receptors were also expressed in in LC, breast cancer, colorectal carcinoma, cervical carcinoma cell lines by RT-PCR assay. The Ach was detectable in LC and breast cancer cell lines or their cultured medium. Therefore, these data strongly suggest that ACh autocrine system may be a common essential signaling system related to growth and survival in various types of cancers.2. Autocrine ACh system correlated to cell growth and anti-apoptosis in HCC and LCBased on data presented above, we examined the biological impact of these ACh autocrine-related molecules. Firstly, we tested the biological effect of AChR agonist as well as antagonist on growth of HCC cells. The nAChR agonist, nicotine (Nic) and the mAChR agonist, carbachol (Carb) could significantly enhance the cancer cell growth; while the nAChR antagonist, mecamylamine (MEC) and the mAChR antagonist, atropine (Atr) inhibited the cell growth. Secondly, we observed the effect of alteration of ACh level on the cell growth. We found that neostigmine (Neo), the inhibitor of AChE could promote the cell growth as well as the transition of cell cycle, particularly the rate of entry into S phase. Furthermore, addition of ACh, Nic and Carb could increase the percentage of S population after nocadazole synchronization. We also found that the addition of recombinant AChE into the culture medium could retard the rate of cell growth. Thirdly, we transfected the siRNA constructs into HCC cells to knock down CHAT expression, resulting in the decrease of cell growth rate. These findings consistently indicate that ACh and its receptor activation are essential of growth of HCC cells.The equally important findings are the effect of ACh autocrine system correlated to cell apoptosis regulation. We found the anti-apoptosis effect on HCC cells treated with adriamycin (ADR) and etopside (VP-16). Nic and Carb had the similar antiapoptotic effect though the latter was less potent than the former. On the contrary but reasonably, the addition of exogenous AChE could enhance the chemotherapy drug-induced apoptosis. Similarly, the knockdown of CHAT by siRNA, the drug-induced apoptosis was enhanced; thus sentizing the cells toward to drug-induced cell death.For further understanding the status and role fo expressed receptor, we examined the a7 nAChR in HCC cell line Bel-7402, HepG2, Hep3B, Huh-7 and lung cancer cell line H446, A549, SPC-A-1, H460 and H1299 cells by a binding assay with a specific antagonistα-Bgt labled with fluorescent dye (FITC-α-Bgt). Among these cell lines, FITC-α-Bgt was positively bound to cells of HCC cell line Bel-7402 and HepG2 and lung cancer cell lines H446, H460 and H1299. We had to mention that the FITC-α-Bgt binding regions on cell surface were distributed in clusters. The clustered pattern of receptors might be putatively suggested as functional receptors usually localized in membrane lipid rafts. Besids, we found thatα-Bgt, MLA (α7 antagonist), MEC and Atrl could block the DNA synthesis in Bel-7402 induced by Nic, although their abilities to cell growth inhibition was very different.Based on the data of these in vitro experiments, we hope to know the effect of ACh system in vivo. By using the Bel-7402 xenograft in nude mouse model, we found that Nic could promote the tumor growth, MLA had no significant effect on tumor growth. Therefore, our data in animal model could confirm that ACh system played an important role in regulation of cell growth in cancer.In addition to studies on HCC cell system, we also found the same role played by ACh autocrine system in LC system. The increase ACh level or Nic treatment could increase the LC cell colony formation rate and enhance the cell growth. The blockage of ACh and nAChR interaction could just produce the opposite effects.Therefore, ACh autocrine system has been proved to be an important system for regulating cell growth and apoptosis both in HCC and LC system, and may be a potential area or drug discovery as well as for design of new therapeutic strategy.3. Activation of ACh system induces a transient calcium influx, and activates MAPK and AKT pathwayExogenous Nic or Ach treatment dose-dependently induced the opening of calcium channel, recorded as a sparkle calcium peak, which could be partially inhibited by MEC, Atr,α-Bgt or AS-α7 treatment. Increased extracellular Ca²⁺ concentration could enhance Ca²⁺ influx, suggesting that increased influx of calcium would induce a cascade of magnification of signal transduction. Similar results were observed in membrane potential alteration in the presence of Nic or Carb. Moreover, voltage-operated calcium channel (VOCC) antagonist nifedipine (Nif) impaired the Nic- and Carb-stimulated Ca²⁺ influx and membrane potential.To understand the possible signal transduction pathway related to ACh autocrie activation process, we studied the phosphoration pattern of MEK/ERK pathway. We identified the phosphorylation of MEK, ERK1/2 and p90^RSK in 5-60min after Nic treatment. The peak of activation was at 15-30min, and faded after about 60 min. Pretreatment with MEC could significantly inhibit the phosphorylation of MEK and ERK1/2 at 30min, while Atr pretreatment did not affect the phosphorylation of these molecules. In al nAChR activated HCC Bel-7402 cells,α-Bgt or AS-α7 treatment could also suppress the effect induced by Nic treatment. More importantly, the increase level of ACh, resulted from AChE inhibitor Neo treatment, could enhance the phosphorylation of MEK, ERK1/2, and p90RSK, while MEC treatment could deprive the effect of Neo treatment. Pretreatment ofα-Bgt or AS-α7 transfection reduced the Nic-induced MAPK pathway activity in Bel-7402. Agonists of ACh system could also activate the AKT, while MEC significantly decrease their effects. Meanwhile, a7 inhibitor could partially block the signal pathway.In conclusion, our study demonstrated the existence of a non-neuronal ACh autocrine system in HCC and LC. Activated ACh autocrine system could induce cell proliferation or apoptosis resistance possibly through calcium influx-triggered activation of MAPK and AKT signal pathway. Therefore, interference of ACh system by inhibiting ACh autocrine, blocking calcium signal, or inhibitory receptor unction, might provide a new prospects in approached in design of new drugs or treatment for HCC and LC. Since ACh autocrine system seems to be uniquitously present in various types of epithelial cancer cells, our finding might put some new insights into understanding of molecular mechanism and control of cancer development.