肝细胞肝癌和肺癌中乙酰胆碱自泌系统的研究
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摘要
原发性肝细胞肝癌(hepatocellular carcinoma，HCC)和肺癌(lung cancer，LC)是恶性程度极高、预后极差的恶性肿瘤。数十年来，在诊断、治疗和预后等各方面的进展十分有限，其根源是这些肿瘤发生发展的确切分子机制所知有限，难以做到真正有针对性的治疗。本实验室前期大规模功能基因筛选中发现了一批与肝癌生长密切相关的基因，就包含一些与神经递质受体相关分子。近期的多项研究表明，多种类型癌症的发生或进展与原神经系统的组成成分的改变密切相关。这些事实提示：癌症的发生和进展可能与神经体系某些组分的改变存在尚不为人知的内在联系。本论文探讨了非神经起源的乙酰胆碱(acetylcholine，ACh)系统在HCC和LC中的合成、分泌及自泌系统相关分子的表达、功能和信号传导，初步明确ACh自泌系统在这两种肿瘤进展中的正调控作用。
1．HCC和LC具有自泌ACh系统
ACh系统是主要的兴奋性神经信号通路，包括ACh，烟碱型受体(nicotinic ACh receptor，nAChR)和毒覃碱型受体(muscrutic ACh receptor，mAChR)，ACh合成酶(choline acetyltransferase，CHAT)，ACh囊泡转运通道(vesicle ACh transporter，VAChT)和ACh降解酶(acetylcholinesterase，AChE)等主要组成分子。在肝癌细胞系、肝癌组织和癌旁肝组织中，通过RT-PCR、Western blot检测到nAChR、mAChR、CHAT和AChE等的基因表达；应用cDNA array和Real time-PCR检测了肝癌组织和配对癌旁组织mRNA表达差异，发现15对肝癌组织中有明显AChE表达下调(P＜0.001)，而在5例人正常肝组织中AChE mRNA表达水平远高于癌旁和癌。经组织芯片证明，与正常和癌旁组织相比，肝癌组织的AChE在蛋白水平上同样呈下降表达，而其它各组分的改变没有相似的显著差异。这些发现提示肝癌可能具有胆碱能表型(cholinergic phenotype)，在癌组织或癌组织微环境中ACh含量可能更高。
我们用不同的物理化学方法定量检测ACh。应用HPLC-MS／MS定量检测了肝癌、LC细胞系的胞内ACh水平；在7对肝癌组织和癌旁组织中ACh含量无统计学差异，影响统计的主要因素可能与标本量大小和标本收集过程中质控等有关。因此，需要更多、质控标准更高的样品以最终确定肝癌和癌旁、正常组织等ACh含量差别是否具有规律性。我们同时使用了灵敏度、特异性更高的检测方法HPLC-ECD，检测到多种肝癌细胞培养液中存在ACh；加入AChE专一性抑制剂neostigmine(Neo)后，培液中ACh水平明显上升(P＜0.05)，由此证明了肝癌细胞可合成并分泌ACh。
同时，我们用RT-PCR方法证实ACh系统关键组分，包括CHAT、VAChT和α7受体等，在LC、乳腺癌等多种不同类型肿瘤细胞系表达。ACh本身还直接在LC细胞内和细胞培液中被定量检测到，乳腺癌细胞和培液中亦被检出。我们的发现表明ACh含量在不同类型肿瘤、同一类型肿瘤不同细胞系之间存在较大差别。据此，我们推测ACh自泌系统可能是所有癌症共同存在的一种新的信号系统。
2．自泌ACh系统诱发HCC和LC生长和凋亡抑制
在确定ACh自泌系统存在于肝、肺癌的前提下，我们进一步考察了该系统对癌细胞和凋亡方面的影响。我们发现nAChR激动剂尼古丁(nicotine，Nic)和mAChR激动剂carbachol(Carb)可显著促进肝癌细胞增殖；nAChR拮抗剂mecamylamine(MEC)和mAChR拮抗剂atropine(Atr)抑制细胞增长；抑制ACh降解酶功能诱导内源性ACh水平增加，能促进肝癌细胞明显增殖，并增加细胞S期比例，此外，细胞经nocodazole同步化后，外源ACh、Nic和Carb均显著增加S期比例；而在细胞培养上清中加入重组AChE，则显著降低了细胞增殖速度；采用siRNA技术knock down ACh合成酶CHAT表达量，可降低细胞增殖速率。
同样重要的发现是，自泌ACh系统活化抑制体外细胞凋亡。Nic对抗化疗药物阿霉素(adriamycin，ADR)和鬼臼毒素(etopside，VP-16)等诱导的细胞凋亡；Carb亦有抗凋亡诱导剂的效果，但药效弱于Nic。反之，外源AChE增强凋亡诱导剂的效果，但本身不能诱导细胞死亡。采用siRNA技术knock down ACh合成酶CHAT，细胞凋亡明显增加，同时可增敏ADR等死亡诱导剂的药效。
我们又进一步观察了α7受体在ACh自泌的肝癌细胞中所起的作用。利用特异性抑制剂FITC-α-bagrotoxin(α-Bgt)进行标记试验，证实肝癌细胞系HepG2和Bel-7402，肺癌H446、H460和H1299细胞均存在功能性α7受体高表达；在四个来自不同个体肝细胞癌的原代培养细胞中，发现有部分细胞可被FITC-α-Bgt标记；值得注意的是，肝癌细胞系或原代培养的肝癌细胞中，FITC-α-Bgt标记后共聚焦实验发现该活性受体能形成cluster现象。α-Bgt、MLA(α7受体拮抗剂)、MEC和Atr可部分阻断Nic诱导的Bel-7402细胞的DNA合成。
在前述体外实验的基础上，我们进行了动物体内实验的观察。通过Bel-7402异种移植瘤模型，发现Nic可促进Bel-7402移植瘤生长，MEC则显著抑制Bel-7402生长，MLA仅有部分抑制作用，明显弱于MEC，不具有统计学意义。上述结果进一步证实了自泌ACh系统可促进肝癌生长的作用。
在肺癌的研究中，发现LC细胞中也存在ACh自泌体系，如增加ACh含量或外加激动剂Nic，可增强克隆形成和加速细胞生长；而阻滞ACh-nAChR的相互作用减缓细胞增殖和克隆形成。上述实验表明，无论HCC或LC，ACh系统激活可显著增强细胞增殖和抗凋亡作用，干扰ACh-AChR互作可能是一种有潜力的抑制肝癌等癌症进展或治疗的新策略。
3．ACh系统活化诱导癌细胞瞬时Ca~(2+)流，增强MAPK和AKT通路活化
肝癌细胞ACh系统活化诱导瞬时Ca~(2+)内流。外源Nic、ACh和Carb的加入，可诱导肝癌细胞内Ca~(2+)通道开放，记录到一个瞬时Ca~(2+)峰，且有显著的剂量依赖效应，加入MEC或Atr可部分抑制该效应；在Bel-7402和HepG2中，α-Bgt和转染α7受体反义寡核苷酸(AS-α7)的细胞的Nic诱导效应明显减弱；电压们控Ca~(2+)阻滞剂nifedipine(Nif)接近完全阻滞Nic作用；增加细胞外Ca~(2+)浓度，可记录更高的Ca~(2+)内流峰，提示增加Ca~(2+)浓度能增强信号传导。
肝癌系统的ACh系统活化同时诱发瞬时膜电位去极化状态。Nic、Carb可激发瞬时膜电位变化，记录到一个瞬时电位去极化峰，用MEC或Atrl预处理可显著减弱峰值。用VOCC抑制剂处理细胞，Nic和Carb不能激发电位去极化，说明去极化过程与ACh系统活化诱导Ca~(2+)内流，打开VOCC有关。
肝癌细胞ACh系统的活化可增强MAPK通路活性。Nic和ACh在5-60 min内有效激活MEK、ERK1／2和p90RSK的磷酸化，15-30分钟左右达到最高，60分钟时明显减弱；MEC预处理后，30分钟时显著减弱MEK、ERK1／2磷酸化，而Atr加入对MEK、ERK1／2的的影响不显著，在60分钟内相关蛋白的表达量无有意义的变化。α7活化的肝癌细胞系Bel-7402中加入α-Bgt或AS-α7转染，均如MEC一样可抑制外源刺激因子Nic和ACh的影响。在Bel-7402细胞中，α-Bgt预处理和AS-α7瞬时转染也可部分减弱该通路活化。
ACh系统活化还可增强AKT通路活性。AKT通路也可被外源性激动剂如Nic激活，MEC预处理可减少AKT磷酸化，α7抑制剂处理Bel-7402细胞可导致该通路活化被部分抑制。
综上所述，我们的研究证明HCC和LC具有非神经来源的ACh自泌系统；ACh系统激活诱发Ca~(2+)内流，增强MAPK和AKT通路，引起细胞增殖和死亡抑制。若干扰ACh信号系统，如减少ACh分泌量、阻断Ca~(2+)信号、抑制受体等，能有效抑制肝癌的进展。ACh自泌体系参与HCC和NSCLC发展过程的一些关键的分子调控和作用，有可能是HCC等肿瘤发生发展中一个新的重要因素。因此，基于调变ACh系统活性，可能是对肝癌和LC治疗的一种潜在的新策略。本研究还进一步提示：ACh自泌系统可能是所有癌症共存的一种新的信号系统，因而对多种恶性肿瘤的防治策略可能有普遍意义。
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 NSCLC
ACh 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 LC
Based 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 pathway
Exogenous 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 Ca~(2+) concentration could enhance Ca~(2+) 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 Ca~(2+) 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.
1．HCC和LC具有自泌ACh系统
ACh系统是主要的兴奋性神经信号通路，包括ACh，烟碱型受体(nicotinic ACh receptor，nAChR)和毒覃碱型受体(muscrutic ACh receptor，mAChR)，ACh合成酶(choline acetyltransferase，CHAT)，ACh囊泡转运通道(vesicle ACh transporter，VAChT)和ACh降解酶(acetylcholinesterase，AChE)等主要组成分子。在肝癌细胞系、肝癌组织和癌旁肝组织中，通过RT-PCR、Western blot检测到nAChR、mAChR、CHAT和AChE等的基因表达；应用cDNA array和Real time-PCR检测了肝癌组织和配对癌旁组织mRNA表达差异，发现15对肝癌组织中有明显AChE表达下调(P＜0.001)，而在5例人正常肝组织中AChE mRNA表达水平远高于癌旁和癌。经组织芯片证明，与正常和癌旁组织相比，肝癌组织的AChE在蛋白水平上同样呈下降表达，而其它各组分的改变没有相似的显著差异。这些发现提示肝癌可能具有胆碱能表型(cholinergic phenotype)，在癌组织或癌组织微环境中ACh含量可能更高。
我们用不同的物理化学方法定量检测ACh。应用HPLC-MS／MS定量检测了肝癌、LC细胞系的胞内ACh水平；在7对肝癌组织和癌旁组织中ACh含量无统计学差异，影响统计的主要因素可能与标本量大小和标本收集过程中质控等有关。因此，需要更多、质控标准更高的样品以最终确定肝癌和癌旁、正常组织等ACh含量差别是否具有规律性。我们同时使用了灵敏度、特异性更高的检测方法HPLC-ECD，检测到多种肝癌细胞培养液中存在ACh；加入AChE专一性抑制剂neostigmine(Neo)后，培液中ACh水平明显上升(P＜0.05)，由此证明了肝癌细胞可合成并分泌ACh。
同时，我们用RT-PCR方法证实ACh系统关键组分，包括CHAT、VAChT和α7受体等，在LC、乳腺癌等多种不同类型肿瘤细胞系表达。ACh本身还直接在LC细胞内和细胞培液中被定量检测到，乳腺癌细胞和培液中亦被检出。我们的发现表明ACh含量在不同类型肿瘤、同一类型肿瘤不同细胞系之间存在较大差别。据此，我们推测ACh自泌系统可能是所有癌症共同存在的一种新的信号系统。
2．自泌ACh系统诱发HCC和LC生长和凋亡抑制
在确定ACh自泌系统存在于肝、肺癌的前提下，我们进一步考察了该系统对癌细胞和凋亡方面的影响。我们发现nAChR激动剂尼古丁(nicotine，Nic)和mAChR激动剂carbachol(Carb)可显著促进肝癌细胞增殖；nAChR拮抗剂mecamylamine(MEC)和mAChR拮抗剂atropine(Atr)抑制细胞增长；抑制ACh降解酶功能诱导内源性ACh水平增加，能促进肝癌细胞明显增殖，并增加细胞S期比例，此外，细胞经nocodazole同步化后，外源ACh、Nic和Carb均显著增加S期比例；而在细胞培养上清中加入重组AChE，则显著降低了细胞增殖速度；采用siRNA技术knock down ACh合成酶CHAT表达量，可降低细胞增殖速率。
同样重要的发现是，自泌ACh系统活化抑制体外细胞凋亡。Nic对抗化疗药物阿霉素(adriamycin，ADR)和鬼臼毒素(etopside，VP-16)等诱导的细胞凋亡；Carb亦有抗凋亡诱导剂的效果，但药效弱于Nic。反之，外源AChE增强凋亡诱导剂的效果，但本身不能诱导细胞死亡。采用siRNA技术knock down ACh合成酶CHAT，细胞凋亡明显增加，同时可增敏ADR等死亡诱导剂的药效。
我们又进一步观察了α7受体在ACh自泌的肝癌细胞中所起的作用。利用特异性抑制剂FITC-α-bagrotoxin(α-Bgt)进行标记试验，证实肝癌细胞系HepG2和Bel-7402，肺癌H446、H460和H1299细胞均存在功能性α7受体高表达；在四个来自不同个体肝细胞癌的原代培养细胞中，发现有部分细胞可被FITC-α-Bgt标记；值得注意的是，肝癌细胞系或原代培养的肝癌细胞中，FITC-α-Bgt标记后共聚焦实验发现该活性受体能形成cluster现象。α-Bgt、MLA(α7受体拮抗剂)、MEC和Atr可部分阻断Nic诱导的Bel-7402细胞的DNA合成。
在前述体外实验的基础上，我们进行了动物体内实验的观察。通过Bel-7402异种移植瘤模型，发现Nic可促进Bel-7402移植瘤生长，MEC则显著抑制Bel-7402生长，MLA仅有部分抑制作用，明显弱于MEC，不具有统计学意义。上述结果进一步证实了自泌ACh系统可促进肝癌生长的作用。
在肺癌的研究中，发现LC细胞中也存在ACh自泌体系，如增加ACh含量或外加激动剂Nic，可增强克隆形成和加速细胞生长；而阻滞ACh-nAChR的相互作用减缓细胞增殖和克隆形成。上述实验表明，无论HCC或LC，ACh系统激活可显著增强细胞增殖和抗凋亡作用，干扰ACh-AChR互作可能是一种有潜力的抑制肝癌等癌症进展或治疗的新策略。
3．ACh系统活化诱导癌细胞瞬时Ca~(2+)流，增强MAPK和AKT通路活化
肝癌细胞ACh系统活化诱导瞬时Ca~(2+)内流。外源Nic、ACh和Carb的加入，可诱导肝癌细胞内Ca~(2+)通道开放，记录到一个瞬时Ca~(2+)峰，且有显著的剂量依赖效应，加入MEC或Atr可部分抑制该效应；在Bel-7402和HepG2中，α-Bgt和转染α7受体反义寡核苷酸(AS-α7)的细胞的Nic诱导效应明显减弱；电压们控Ca~(2+)阻滞剂nifedipine(Nif)接近完全阻滞Nic作用；增加细胞外Ca~(2+)浓度，可记录更高的Ca~(2+)内流峰，提示增加Ca~(2+)浓度能增强信号传导。
肝癌系统的ACh系统活化同时诱发瞬时膜电位去极化状态。Nic、Carb可激发瞬时膜电位变化，记录到一个瞬时电位去极化峰，用MEC或Atrl预处理可显著减弱峰值。用VOCC抑制剂处理细胞，Nic和Carb不能激发电位去极化，说明去极化过程与ACh系统活化诱导Ca~(2+)内流，打开VOCC有关。
肝癌细胞ACh系统的活化可增强MAPK通路活性。Nic和ACh在5-60 min内有效激活MEK、ERK1／2和p90RSK的磷酸化，15-30分钟左右达到最高，60分钟时明显减弱；MEC预处理后，30分钟时显著减弱MEK、ERK1／2磷酸化，而Atr加入对MEK、ERK1／2的的影响不显著，在60分钟内相关蛋白的表达量无有意义的变化。α7活化的肝癌细胞系Bel-7402中加入α-Bgt或AS-α7转染，均如MEC一样可抑制外源刺激因子Nic和ACh的影响。在Bel-7402细胞中，α-Bgt预处理和AS-α7瞬时转染也可部分减弱该通路活化。
ACh系统活化还可增强AKT通路活性。AKT通路也可被外源性激动剂如Nic激活，MEC预处理可减少AKT磷酸化，α7抑制剂处理Bel-7402细胞可导致该通路活化被部分抑制。
综上所述，我们的研究证明HCC和LC具有非神经来源的ACh自泌系统；ACh系统激活诱发Ca~(2+)内流，增强MAPK和AKT通路，引起细胞增殖和死亡抑制。若干扰ACh信号系统，如减少ACh分泌量、阻断Ca~(2+)信号、抑制受体等，能有效抑制肝癌的进展。ACh自泌体系参与HCC和NSCLC发展过程的一些关键的分子调控和作用，有可能是HCC等肿瘤发生发展中一个新的重要因素。因此，基于调变ACh系统活性，可能是对肝癌和LC治疗的一种潜在的新策略。本研究还进一步提示：ACh自泌系统可能是所有癌症共存的一种新的信号系统，因而对多种恶性肿瘤的防治策略可能有普遍意义。
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 NSCLC
ACh 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 LC
Based 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 pathway
Exogenous 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 Ca~(2+) concentration could enhance Ca~(2+) 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 Ca~(2+) 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.
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