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Cigarette smoking is the leading risk factor for the development of lung cancer. It is estimated that smoking is associated with 80-90% of lung cancer cases throughout the world (see References 1 and 2). The addictive component of cigarette smoke is nicotine. Our published data shows that nicotine promotes the production of acetylcholine (ACh) in human bronchioalveolar carcinoma cells (BACs) (Lau et al., 2013). ACh functions as a growth factor in human BACs. The following protocol is based on a published protocol by (Song et al., 2003), with some modifications (Lau et al., 2013; Song et al., 2008; Song et al., 2003; Sekhon et al., 2003). An important point to remember is that fetal bovine serum (FBS) contains a high amount of acetylcholine (ACh). Therefore, cells must be cultured in serum-free medium to measure ACh in the culture supernatant. Two aliquots of the culture supernatant are used for analysis. This protocol measures the total choline in the cell supernatent under two conditions: 1) After treatment with acetylcholinesterase (AChE), which converts the ACh to choline (also called the total choline sample) and 2) after measuring the amount of free choline in the sample. The concentration of ACh in the sample calculated by subtracting the free choline from the total choline.

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Measurement of Acetylcholine from Cell Lines
细胞系乙酰胆碱的测定

癌症生物学 > 细胞能量学 > 细胞生物学试验 > 新陈代谢
作者: Jamie K. Lau
Jamie K. LauAffiliation: Department of Pharmacology, Physiology, and Toxicology, Marshall University, Huntington, USA
Bio-protocol author page: a1084
Kathleen C. Brown
Kathleen C. BrownAffiliation: Department of Pharmacology, Physiology, and Toxicology, Marshall University, Huntington, USA
Bio-protocol author page: a1085
 and Piyali Dasgupta
Piyali DasguptaAffiliation: Department of Pharmacology, Physiology, and Toxicology, Marshall University, Huntington, USA
For correspondence: dasgupta@marshall.edu
Bio-protocol author page: a1086
Vol 3, Iss 24, 12/20/2013, 3708 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1007

[Abstract] Cigarette smoking is the leading risk factor for the development of lung cancer. It is estimated that smoking is associated with 80-90% of lung cancer cases throughout the world (see References 1 and 2). The addictive component of cigarette smoke is nicotine. Our published data shows that nicotine promotes the production of acetylcholine (ACh) in human bronchioalveolar carcinoma cells (BACs) (Lau et al., 2013). ACh functions as a growth factor in human BACs. The following protocol is based on a published protocol by (Song et al., 2003), with some modifications (Lau et al., 2013; Song et al., 2008; Song et al., 2003; Sekhon et al., 2003). An important point to remember is that fetal bovine serum (FBS) contains a high amount of acetylcholine (ACh). Therefore, cells must be cultured in serum-free medium to measure ACh in the culture supernatant. Two aliquots of the culture supernatant are used for analysis. This protocol measures the total choline in the cell supernatent under two conditions: 1) After treatment with acetylcholinesterase (AChE), which converts the ACh to choline (also called the total choline sample) and 2) after measuring the amount of free choline in the sample. The concentration of ACh in the sample calculated by subtracting the free choline from the total choline.

[Abstract]

Materials and Reagents

  1. A549 cells (American Type Culture Collection)
  2. Human Epidermal Growth Factor (EGF) (Sigma Chemical, catalog number: E9644 )
  3. 100x Insulin Transferrin Selenium (ITS) (Life Technologies, catalog number: 41400-045 )
  4. Rosewell Park Memorial Institute (RPMI) Medium -1640 (ATCC, catalog number: 41400-045)
  5. 50 μM Hydrocortisone (Sigma-Aldrich, catalog number: H6909 )
  6. Boveine serum albumin (BSA) (US Biochem, catalog number: 10857 )
  7. Disposable sterile tissue culture filters (Corning, catalog number: 431161 )
  8. Choline/acetylcholine Quantification Kit (BioVision, catalog number: K615-100 )
  9. Liquid nitrogen
  10. Serum-Free RPMI (SF-RPMI) (see Recipes)
  11. Neostigmine (Sigma Chemical, catalog number: N2001 ) (see Recipes)

Equipment

  1. 60 mm cell culture dishes (Corning, catalog number: 353002 )
  2. Microfuge tube
  3. ELISA Reader
  4. Lyophilizer (Labonco)
  5. Centrifuge
  6. CO2 cell culture incubator
  7. -80 °C freezer

Procedure

  1. A549 cells were grown in 5ml serum-free RPMI (SF-RPMI) to 90%-95% confluence in 60 mm cell culture dishes in a cell culture incubator set at 5% CO2 and 37 °C (Lau et al., 2013; Song et al., 2008; Song et al., 2003).
  2. On the day of the assay, 100 μM neostigmine (a chemical inhibitor of AChE in cells) was added to each plate for four hours at 37 °C. The plate contained 3 ml of media. Please see Notes section about optimization of the concentration of neostigmine.
  3. Four hours after the addition of neostigmine, the relevant concentration of test compund (which promotes/inhibits the secretion of ACh) was added and the cells were incubated at 37 °C for 36 h. An example of a compound which promotes the production of ACh is 100 nM nicotine.
  4. The supernatant (medium) was collected and spun at 800 x g.
  5. The supernatants were frozen at -80 °C and then lyophilized. The lyophilizer was set to a pressure of 10 micron Hg or below that value. The samples were lyophilized overnight.
  6. Subsequently, the lyophilizate was reconstituted with 1/5 volume autoclaved water (600 μl autoclaved water), snap frozen in liquid nitrogen and stored at -80 °C until further analysis.
  7. The amount of ACh in the sample was measured using the Choline/acetylcholine Quantification Kit, according to manufacturer’s instructions (http://www.biovision.com/choline-acetylcholine-quantification-colorimetric-fluorometric-kit-2910.html). The protocol outlined in the assay kit will be attached here. Each sample was assayed in triplicate.

Notes

ACh is secreted by lung cancer cells into the extracellular environment. Part of the ACh binds to nicotinic acetylcholine receptors and muscarinic acetylcholine receptors on the same lung cancer cells stimulating their proliferation in an autocrine manner. The excess ACh is quickly degraded by the enzyme AChE to generate choline which is then taken up by the cells to synthesize new ACh. Therefore, it is essential to inhibit the AChE to measure the ACh produced by the lung cancer cells.
Optimization of the concentration of neostigmine:

  1. Neostigmine is added to inhibit the enzyme AChE in cells. It is important to titrate the amount of neostigmine to be used otherwise it may compromise the readout of the assay.
  2. A549 cells were grown in serum-free RPMI (SF-RPMI) to 90%-95% confluence in 60 mm cell culture dishes in a cell culture incubator set at 5% CO2  and 37 °C.
  3. On the day of the assay, the following concentrations of neostigmine were added
    1. 10 μM
    2. 20 μM
    3. 50 μM
    4. 100 μM
    5. 200 μM
    6. 300 μM
  4. Each plate contained 3 ml of media. The cells were incubated at 37 °C for 36 h.
  5. Follow the steps 4-7 of the Procedure section described above.
  6. In our experiments, the production of ACh varied with increasing concentrations of neostigmine as shown below in Figure 1A. Figure 1B shows that the amount of AChE in all three cell lines is similar. The baseline amount of ACh is highest in the presence of 100-200 uM neostigmine. Therefore, we selected 100 uM neostigmine for all our experiments.  It is probable that 300 uM of neostigmine is toxic so the cells and causes cell death so the levels of ACh are lower. 


    Figure 1. Optimization of the concentration of neostigmine for measurement of ACh from A549 human lung cancer cells. (A) A549 human lung cancer cells were cultured in SF-RPMI and the amount of ACh produced was measured in the presence of varying concentrations of neostigmine.   The baseline amount of ACh is highest in the presence of 100-200 uM neostigmine. (B) Western blotting analysis shows the presence of robust amounts of AChE in three human lung cancer cell lines namely A549, H358 and H650.  Values indicated by the * are statistically significant relative to controls (*P<0.05).

Recipes

  1. Serum-Free RPMI (SF-RPMI), 100 ml
    To 50 ml of RPMI in a sterile bottle, add the following:
    100 mg BSA to and stir to dissolve
    1 ml ITS
    100 ul of 50 uM hydrocortisone
    1 mg EGF
    Make up the volume to 100 ml with RPMI
    Filter sterilize using a 0.22 μm filter
    Stored at 4 °C
  2. Neostigmine (Stock = 100 mM)
    Weigh 33.4 mg of neostigmine in a sterile microfuge tube
    Dissolve it in autoclaved water
    Aliquot in multiple microfuge tubes
    Stored at -20 °C

Acknowledgments

We would like to acknowledge the following publications on which this protocol is based: Song et al. (2008); Song et al. (2003a); Song et al. (2003b). We thank Dr. Srikumar Chellappan and his laboratory for their continuous support. This work was supported by the grants Young Clinical Scientist Award (#82115) from the Flight Attendant Medical Association, Miami, FL and 1R15CA161491-01A1 from NIH to PDG. KCB is a recipient of a graduate fellowship from the WVSGC.

References

  1. Cancer, I.A.R.C. (2004). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. In Tobacco Smoke and Involuntary Smoking: IARC, Lyon, France. 51-1187.
  2. Lau, J. K., Brown, K. C., Thornhill, B. A., Crabtree, C. M., Dom, A. M., Witte, T. R., Hardman, W. E., McNees, C. A., Stover, C. A., Carpenter, A. B., Luo, H., Chen, Y. C., Shiflett, B. S. and Dasgupta, P. (2013). Inhibition of cholinergic signaling causes apoptosis in human bronchioalveolar carcinoma. Cancer Res 73(4): 1328-1339.
  3. Song, P., Sekhon, H. S., Fu, X. W., Maier, M., Jia, Y., Duan, J., Proskosil, B. J., Gravett, C., Lindstrom, J., Mark, G. P., Saha, S. and Spindel, E. R. (2008). Activated cholinergic signaling provides a target in squamous cell lung carcinoma. Cancer Res 68(12): 4693-4700.
  4. Song, P., Sekhon, H. S., Jia, Y., Keller, J. A., Blusztajn, J. K., Mark, G. P. and Spindel, E. R. (2003). Acetylcholine is synthesized by and acts as an autocrine growth factor for small cell lung carcinoma. Cancer Res 63(1): 214-221.
  5. Song, P., Sekhon, H. S., Proskocil, B., Blusztajn, J. K., Mark, G. P. and Spindel, E. R. (2003). Synthesis of acetylcholine by lung cancer. Life Sci 72(18-19): 2159-2168.
  6. The HEALTH Consequences of Smoking. (2004). A report of the Surgeon General's Office on Smoking and Health, DHHS, Washington DC.

材料和试剂

  1. A549细胞(美国典型培养物保藏中心)
  2. 人表皮生长因子(EGF)(Sigma Chemical,目录号:E9644)
  3. 100x胰岛素转铁蛋白硒(ITS)(Life Technologies,目录号:41400-045)
  4. Rosewell Park Memorial Institute(RPMI)Medium-1640(ATCC,目录号:41400-045)
  5. 50μM氢化可的松(Sigma-Aldrich,目录号:H6909)
  6. 牛血清白蛋白(BSA)(US Biochem,目录号:10857)
  7. 一次性无菌组织培养过滤器(Corning,目录号:431161)
  8. 胆碱/乙酰胆碱定量试剂盒(BioVision,目录号:K615-100)
  9. 液氮
  10. 无血清RPMI(SF-RPMI)(参见配方)
  11. 炔雌醇(Sigma Chemical,目录号:N2001)(参见Recipes)

设备

  1. 60mm细胞培养皿(Corning,目录号:353002)
  2. 微杯管
  3. ELISA读数器
  4. 冻干机(Labonco)
  5. 离心机
  6. CO 2细胞培养孵化器
  7. -80°C冰箱

程序

  1. 将A549细胞在设定为5%CO 2和37℃的细胞培养箱中在60mm细胞培养皿中在5ml无血清RPMI(SF-RPMI)中生长至90%-95%汇合 (Lau等人,2013; Song等人,2008; Song等人,2003)。
  2. 在测定当天,在37℃下将100μM新斯的明(细胞中的AChE的化学抑制剂)加入每个板中4小时。 该板含有3ml培养基。 请参见注释部分关于新斯的明浓度的优化
  3. 加入新斯的明4小时后,加入相应浓度的测试化合物(其促进/抑制ACh的分泌),并将细胞在37℃下孵育36小时。促进ACh产生的化合物的实例是100nM尼古丁。
  4. 收集上清液(培养基)并以800×g离心
  5. 将上清液在-80℃冷冻,然后冻干。将冻干机设定为10微米Hg的压力或低于该值。将样品冻干过夜。
  6. 随后,将冻干物用1/5体积的高压灭菌水(600μl高压灭菌水)重构,在液氮中快速冷冻并储存在-80℃直至进一步分析。
  7. 使用胆碱/乙酰胆碱定量试剂盒,根据制造商的说明书测量样品中的ACh的量( http://www.biovision.com/choline-acetylcholine-quantification-colorimetric-fluorometric-kit-2910.html )。测定试剂盒中概述的方案将附于此处。每个样品一式三份进行测定

笔记

ACh由肺癌细胞分泌到细胞外环境中。部分ACh与同样的肺癌细胞上的烟碱乙酰胆碱受体和毒蕈碱乙酰胆碱受体结合,以自分泌方式刺激它们的增殖。过量的ACh被酶AChE快速降解以产生胆碱,然后细胞吸收所述胆碱以合成新的ACh。因此,必须抑制AChE以测量肺癌细胞产生的ACh 新斯的明浓度的优化:

  1. 加入新斯的明以抑制细胞中的酶AChE。重要的是滴定要使用的新斯的明的量,否则其可能损害测定的读出。
  2. A549细胞在设定为5%CO 2的细胞培养箱中在60mm细胞培养皿中在无血清RPMI(SF-RPMI)中生长至90%-95%汇合。和37℃。
  3. 在测定当天,加入以下浓度的新斯的明
    1. 10μM
    2. 20μM
    3. 50μM
    4. 100μM
    5. 200μM
    6. 300μM
  4. 每个平板含有3ml培养基。将细胞在37℃孵育36小时。
  5. 按照上述步骤的步骤4-7。
  6. 在我们的实验中,ACh的产生随新斯的明浓度的增加而变化,如图1A所示。图1B显示所有三种细胞系中的AChE量相似。在100-200uM新斯的明存在下,ACh的基线量最高。因此,我们选择100 uM新斯的明用于所有我们的实验。可能300uM的新斯的明是有毒的,所以细胞和引起细胞死亡,所以ACh的水平较低。


    图1.用于测量来自A549人肺癌细胞的ACh的新斯的明的浓度的优化。 (A)在SF-RPMI中培养A549人肺癌细胞,并在不同浓度的新斯的明存在下测量产生的ACh量。在100-200uM新斯的明存在下,ACh的基线量最高。 (B)Western印迹分析显示在三种人肺癌细胞系即A549,H358和H650中存在稳定量的AChE。由*表示的值相对于对照具有统计学显着性(* P <0.05)。

食谱

  1. 无血清RPMI(SF-RPMI),100ml
    向无菌瓶中的50ml RPMI中加入以下物质:
    100mg BSA,搅拌溶解
    1 ml ITS
    100μl50μM氢化可的松
    1 mg EGF
    用RPMI
    将体积补至100 ml 使用0.22μm过滤器过滤灭菌
    储存在4°C
  2. 炔诺酮(储存= 100mM)
    在无菌微量离心管中称重33.4mg新斯的明 将其溶于高压灭菌水中
    在多个微量离心管中分装
    储存于-20°C

致谢

我们要感谢本协议所基于的以下出版物:Song 等人(2008); Song 等人(2003a); Song等人(2003b)。 我们感谢Srikumar Chellappan博士及其实验室的持续支持。 这项工作得到了来自美国佛罗里达州迈阿密的航空助理医学协会的青年临床科学家奖(#82115),以及从NIH到PDG的1R15CA161491-01A1的支持。 KCB是WVSGC研究生奖学金的接受者。

参考文献

  1. Cancer,I.A.R.C. (2004)。 IARC关于评估人类致癌风险的专着。 In烟雾和非自愿吸烟:IARC,里昂,法国。 51-1187。
  2. Lau,JK,Brown,KC,Thornhill,BA,Crabtree,CM,Dom,AM,Witte,TR,Hardman,WE,McNees,CA,Stover,CA,Carpenter,AB,Luo,H.,Chen,YC,Shiflett ,BS和Dasgupta,P。(2013)。 抑制胆碱能信号传导引起人支气管肺泡癌的凋亡。 /em> 73(4):1328-1339。
  3. Song,P.,Sekhon,HS,Fu,XW,Maier,M.,Jia,Y.,Duan,J.,Proskosil,BJ,Gravett,C.,Lindstrom,J.,Mark,GP,Saha,和Spindel,ER(2008)。 激活的胆碱能信号传导在鳞状细胞肺癌中提供了一个靶点。 68(12):4693-4700。
  4. Song,P.,Sekhon,H.S.,Jia,Y.,Keller,J.A.,Blusztajn,J.K.,Mark,G.P.and Spindel,E.R。(2003)。 乙酰胆碱由小细胞肺癌合成,并作为小细胞肺癌的自分泌生长因子。 Cancer Res 63(1):214-221。
  5. Song,P.,Sekhon,H.S.,Proskocil,B.,Blusztajn,J.K.,Mark,G.P.and Spindel,E.R。(2003)。 肺癌对乙酰胆碱的合成 生命科学 (18-19):2159-2168。
  6. 吸烟的健康后果。 (2004)。卫生署吸烟与健康办公室,DHHS,华盛顿特区的报告
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How to cite this protocol: Lau, J. K., Brown, K. C. and Dasgupta, P. (2013). Measurement of Acetylcholine from Cell Lines. Bio-protocol 3(24): e1007. DOI: 10.21769/BioProtoc.1007; Full Text



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