低氘水對(duì)人肺癌細(xì)胞A549增殖和分化的影響及機(jī)制

戚冰雪，李子宜，叢峰松

(上海交通大學(xué)生命科學(xué)技術(shù)學(xué)院，上海200240)

·論著·

低氘水對(duì)人肺癌細(xì)胞A549增殖和分化的影響及機(jī)制

戚冰雪，李子宜，叢峰松

(上海交通大學(xué)生命科學(xué)技術(shù)學(xué)院，上海200240)

目的 研究低氘水(DDW)對(duì)人肺癌A549細(xì)胞增殖和分化的影響，并探討其作用機(jī)制。方法 取A549細(xì)胞分為觀察組和對(duì)照組，分別用氘含量為50 ppm的DDW配制的培養(yǎng)基、正常水配制的培養(yǎng)基培養(yǎng)30 d，倒置顯微鏡下觀察兩組細(xì)胞形態(tài)，用CCK-8法測(cè)定48 h內(nèi)細(xì)胞生長(zhǎng)情況。觀察組和對(duì)照組細(xì)胞先分別在無(wú)血清的DDW培養(yǎng)基和正常水培養(yǎng)基中培養(yǎng)48 h使細(xì)胞生長(zhǎng)同步化，然后分別加入含10%血清的DDW培養(yǎng)基和正常水培養(yǎng)基繼續(xù)培養(yǎng)，用碘化丙啶染色和流式細(xì)胞分析儀測(cè)定兩組36、48 h時(shí)處于細(xì)胞周期G1、S和G2/M期的細(xì)胞比例；取兩組細(xì)胞裂解液，抽提細(xì)胞總蛋白，Western blot法檢測(cè)絲裂原活化的細(xì)胞外信號(hào)調(diào)節(jié)激酶(MEK1)、表皮生長(zhǎng)因子受體(EGFR)表達(dá)，MEK1第217和第298位絲氨酸磷酸化水平，EGFR第1016和第1110位酪氨酸磷酸化水平；比較兩組肺泡Ⅰ型上皮細(xì)胞特異性蛋白標(biāo)志物AQP5、T1a和肺泡Ⅱ型上皮細(xì)胞特異性蛋白標(biāo)志物SPB、SPC1、SPC2、CFTR的表達(dá)。結(jié)果 對(duì)照組細(xì)胞呈立方形、細(xì)胞間緊密接觸和堆積生長(zhǎng)，觀察組細(xì)胞呈紡錘形、細(xì)胞伸長(zhǎng)、細(xì)胞間接觸較松散。觀察組36、48 h時(shí)的OD值均較對(duì)照組降低(P均<0.05)。血清饑餓后再刺激36 h，觀察組處于G1期的細(xì)胞比例為71.75%，高于對(duì)照組的57.01%；觀察組處于S期的細(xì)胞比例為22.43%，低于對(duì)照組的35.88%(P均<0.01);48 h時(shí)兩組細(xì)胞周期比例比較差異無(wú)統(tǒng)計(jì)學(xué)意義。兩組MEK1、EGFR表達(dá)及EGFR第1016位和第1110位酪氨酸磷酸化水平比較差異無(wú)統(tǒng)計(jì)學(xué)意義，觀察組MEK1第217位和第298位絲氨酸磷酸化的水平降低(P均<0.01)。觀察組SPC2水平較對(duì)照組升高(P<0.01)。結(jié)論 A549細(xì)胞用DDW長(zhǎng)期培養(yǎng)，可顯著抑制其細(xì)胞增殖和分化，其機(jī)制可能與降低MEK1的磷酸化水平、阻滯細(xì)胞周期G1/S期轉(zhuǎn)變以及誘導(dǎo)A549細(xì)胞由Ⅱ型肺泡上皮細(xì)胞向Ⅰ型肺泡上皮細(xì)胞轉(zhuǎn)化、提高細(xì)胞分化程度有關(guān)。

肺癌;低氘水；細(xì)胞周期；絲裂原活化的細(xì)胞外信號(hào)調(diào)節(jié)激酶；細(xì)胞分化

氘是氫的三種同位素之一[1]，自然存在的氘為正常細(xì)胞生長(zhǎng)所必需[2，3]。氘的自然含量因在地球上的采樣位置不同而不同，其濃度范圍為120～160 ppm[4]。氫在地球上以水的形式存在最多，氘含量低于150 ppm的水稱為低氘水(DDW)。DDW可以在體外和體內(nèi)抑制癌細(xì)胞生長(zhǎng)[5，6]，已有報(bào)道將其用于肺癌腦轉(zhuǎn)移患者的輔助治療，使用DDW結(jié)合常規(guī)臨床治療可以延長(zhǎng)腫瘤患者的生存期[7]。表皮生長(zhǎng)因子受體(EGFR)和絲裂原活化的細(xì)胞外信號(hào)調(diào)節(jié)激酶(MEK)通路是影響腫瘤細(xì)胞增殖的重要信號(hào)通路，信號(hào)通路的激活與活性位點(diǎn)的磷酸化水平呈正相關(guān)[8]。2016年6～9月，我們使用DDW培養(yǎng)人肺腺癌A549細(xì)胞，觀察A549細(xì)胞EGFR和MEK1活性位點(diǎn)的磷酸化水平，并檢測(cè)肺泡Ⅰ型上皮細(xì)胞特異性蛋白標(biāo)志物AQP5、T1a和肺泡Ⅱ型上皮細(xì)胞特異性蛋白標(biāo)志物SPB、SPC1、SPC2、CFTR的表達(dá)變化，探討DDW對(duì)人肺腺癌A549細(xì)胞增殖和分化的影響及機(jī)制。

1 材料與方法

1.1 材料 A549細(xì)胞系購(gòu)自中科院上海細(xì)胞庫(kù)，F(xiàn)12K培養(yǎng)基、胎牛血清和0.25%的胰酶-EDTA購(gòu)自Thermo Fisher公司，碘化丙啶、BCA蛋白質(zhì)濃度測(cè)定試劑盒和RIPA緩沖液購(gòu)自碧云天生物技術(shù)有限公司，CCK-8試劑盒購(gòu)自北京博爾邁生物技術(shù)有限公司，DDW(氘含量50 ppm)購(gòu)自上海齊天生物科技有限公司，用于檢測(cè)MEK1、EGFR總蛋白的抗體、抗MEK1第217位絲氨酸磷酸化(MEK-pSer217)和第217位絲氨酸磷酸化(MEK1-pSer298)的抗體以及抗EGFR第1016位酪氨酸磷酸化(EGFR-pTyr1016)和第1110位酪氨酸磷酸化(EGFR-pTyr1110)的抗體購(gòu)自美國(guó)Cell Signaling公司，抗AQ5、T1a、SPC1、SPC2和GADPH抗體購(gòu)自美國(guó)Abcam公司，抗CFTR和SPB抗體購(gòu)自美國(guó)Merk Millipore公司，耦聯(lián)有辣根過(guò)氧化物酶(HRP)的二抗購(gòu)自美國(guó)KPL公司，化學(xué)發(fā)光試劑購(gòu)自美國(guó)GE公司，其他化學(xué)試劑購(gòu)自美國(guó)Sigma公司。

1.2 細(xì)胞分組與處理 將A549細(xì)胞分為對(duì)照組和觀察組，分別加入正常水配制的F12K培養(yǎng)基、DDW配制的F12K培養(yǎng)基中，加入終濃度為2 mmol/L的谷氨酰胺和10%胎牛血清。37 ℃、5% CO2、飽和濕度培養(yǎng)30 d。每天在倒置顯微鏡下觀察細(xì)胞形態(tài)。

1.3 細(xì)胞增殖觀察 將培養(yǎng)30 d的細(xì)胞加入0.25%的胰酶-EDTA消化，以1×105/mL接種于96孔培養(yǎng)板中。于12、24、36、48 h分別加入CCK-8試劑10 μL，繼續(xù)培養(yǎng)顯色90 min，用酶標(biāo)儀在450 nm波長(zhǎng)處測(cè)定吸光度OD值。每個(gè)時(shí)間點(diǎn)設(shè)3個(gè)復(fù)孔，實(shí)驗(yàn)共重復(fù)3次，取平均值。

1.4 細(xì)胞周期分析 將培養(yǎng)30 d的細(xì)胞加入0.25%的胰酶-EDTA消化，以1×105/mL接種到6孔培養(yǎng)板中。細(xì)胞貼壁后，對(duì)照組和觀察組細(xì)胞分別在不含血清的正常水培養(yǎng)基和DDW培養(yǎng)基中饑餓48 h使細(xì)胞生長(zhǎng)同步化，然后加入含10%胎牛血清的正常水培養(yǎng)基和DDW培養(yǎng)基繼續(xù)培養(yǎng)36 h和48 h。加入胰酶消化，將細(xì)胞以2×105/mL重懸于碘化丙錠染色液中，避光染色30 min。用BD公司的FACS CaliburTM進(jìn)行單個(gè)細(xì)胞內(nèi)DNA含量的分析，細(xì)胞群體在細(xì)胞周期不同時(shí)相的分布用ModFit LT軟件進(jìn)行擬合分析。

1.5 EGFR、MEK1蛋白表達(dá)及活性位點(diǎn)磷酸化檢測(cè) 采用Western blot方法。將培養(yǎng)30 d的細(xì)胞用含有蛋白酶抑制劑和磷酸脂酶抑制劑的預(yù)冷PBS洗滌，加入RIPA裂解液反復(fù)吹打裂解細(xì)胞，4 ℃離心收集上清液，用BCA試劑盒測(cè)定總蛋白濃度。每個(gè)泳道上蛋白質(zhì)樣品50 μg進(jìn)行SDS-PAGE后用濕轉(zhuǎn)法將蛋白條帶轉(zhuǎn)移到PVDF膜上。用含4%脫脂奶粉的PBST封閉，加入相應(yīng)抗體(EGFR、MEK1抗體或針對(duì)活性位點(diǎn)磷酸化的抗體)4 ℃一抗孵育過(guò)夜，PBST洗滌3次后，用耦聯(lián)有HRP的二抗(1∶10 000)室溫孵育1 h，PBST洗滌，化學(xué)發(fā)光底物顯色，化學(xué)發(fā)光掃描儀記錄蛋白表達(dá)或蛋白活性位點(diǎn)磷酸化的條帶。以目標(biāo)蛋白與內(nèi)參蛋白GADPH灰度值的比值計(jì)算目標(biāo)蛋白的相對(duì)表達(dá)量。

1.6 肺泡上皮細(xì)胞特異性蛋白標(biāo)志物檢測(cè) 按照1.5中的方法提取兩組細(xì)胞的總蛋白，電泳并轉(zhuǎn)膜后進(jìn)行Western blot分析，用抗AQ5、T1a、CFTR、SPB、SPC1、SPC2的抗體檢測(cè)各蛋白標(biāo)志物的表達(dá)。以目標(biāo)蛋白與內(nèi)參蛋白GADPH灰度值的比值計(jì)算目標(biāo)蛋白的相對(duì)表達(dá)量。

2 結(jié)果

2.1 兩組細(xì)胞形態(tài)比較 對(duì)照組細(xì)胞在倒置顯微鏡下呈立方形，細(xì)胞間接觸緊密和堆積性生長(zhǎng)；觀察組細(xì)胞呈紡錘形，細(xì)胞伸長(zhǎng)，細(xì)胞之間的接觸較對(duì)照組松散。

2.2 兩組細(xì)胞增殖情況比較 觀察組12、24、36、48 h的OD值分別為0.33±0.12、0.38±0.01、0.42±0.02、0.50±0.06；對(duì)照組分別為0.33±0.02、0.37±0.01、0.47±0.05、0.65±0.05。觀察組36、48 h時(shí)的OD值較對(duì)照組降低(P均<0.05)。

2.3 兩組細(xì)胞周期分布比較 觀察組36 h時(shí)處于G1期的細(xì)胞比例高于對(duì)照組，處于S期的細(xì)胞比例低于對(duì)照組(P均<0.01)。48 h時(shí)兩組細(xì)胞周期比例比較差異無(wú)統(tǒng)計(jì)學(xué)意義。見(jiàn)表1。

表1 兩組細(xì)胞周期分布比較

注：與對(duì)照組比較，*P<0.01。

2.4 兩組MEK1、EGFR磷酸化水平比較 觀察組MEK1蛋白表達(dá)與對(duì)照組比較無(wú)統(tǒng)計(jì)學(xué)差異;與對(duì)照組比較，觀察組第217位、第298位絲氨酸的磷酸化水平顯著下調(diào)。觀察組EGFR蛋白表達(dá)與對(duì)照組比較無(wú)統(tǒng)計(jì)學(xué)差異；第1016、第1110位酪氨酸的磷酸化水平也無(wú)明顯變化。

表2 兩組MEK1、EGFR及其活性位點(diǎn)的磷酸化水平

2.5 兩組肺泡上皮特異性標(biāo)志物水平比較 DDW培養(yǎng)30 d后，觀察組AQP5、T1a、SPC1、SPC2水平較對(duì)照組升高，CFTR、SPB水平無(wú)明顯變化。提示觀察組部分A549細(xì)胞的分化類型由Ⅱ型上皮細(xì)胞向Ⅰ型上皮細(xì)胞轉(zhuǎn)化。見(jiàn)表3。

3 討論

表3 兩組肺泡上皮特異性標(biāo)志物水平比較±s)

研究顯示，氘能夠影響細(xì)胞生長(zhǎng)[8]。已有報(bào)道顯示，DDW能顯著延長(zhǎng)前列腺癌和未轉(zhuǎn)移肺癌患者的生存期[5，9]。然而DDW對(duì)腫瘤細(xì)胞生長(zhǎng)抑制的分子機(jī)制目前還不清楚。細(xì)胞周期是細(xì)胞分裂經(jīng)歷的全過(guò)程，S期DNA進(jìn)行復(fù)制，M期細(xì)胞進(jìn)行分裂，S期和M期之前分別為G1生長(zhǎng)期和G2生長(zhǎng)期[10]。本研究顯示，DDW能夠抑制A549細(xì)胞從G1期向S期轉(zhuǎn)化，從而抑制其生長(zhǎng)，這種生長(zhǎng)抑制效應(yīng)是由于DDW阻滯細(xì)胞周期由G1期向G2期轉(zhuǎn)變所致。

研究證明，A549細(xì)胞攜帶致癌基因KRAS/G12S激活突變，這一突變上調(diào)MAPK/ERK信號(hào)通路，從而促進(jìn)A549細(xì)胞的分裂增殖[11，12]。為了探究DDW對(duì)A549細(xì)胞生長(zhǎng)抑制作用的信號(hào)通路，我們選擇了與細(xì)胞有絲分裂密切相關(guān)的兩個(gè)重要分子EGFR和MEK1上與其活性有關(guān)的關(guān)鍵磷酸化位點(diǎn)上磷酸化水平變化進(jìn)行研究。結(jié)果表明，DDW培養(yǎng)對(duì)EGFR的表達(dá)及其第1016及第1110位酪氨酸活性位點(diǎn)的磷酸化水平無(wú)明顯影響；對(duì)MEK1的表達(dá)水平雖無(wú)明顯影響，但可顯著降低其第217位和第298位絲氨酸的磷酸化水平。說(shuō)明DDW可通過(guò)下調(diào)MEK/ERK信號(hào)通路抑制A549細(xì)胞增殖。

肺泡上皮根據(jù)形態(tài)和功能分為Ⅰ型上皮和Ⅱ型上皮，Ⅰ型上皮主要負(fù)責(zé)氣體交換，Ⅱ型上皮通過(guò)分泌表面活性蛋白來(lái)維持肺泡的表面張力[9]。A549細(xì)胞屬于Ⅱ型肺泡上皮細(xì)胞，能夠產(chǎn)生表面活性蛋白[9，13]。本研究發(fā)現(xiàn)，用DDW培養(yǎng)基培養(yǎng)A549細(xì)胞30 d后，細(xì)胞發(fā)生了顯著的形態(tài)學(xué)變化，由原來(lái)的立方形、細(xì)胞間接觸緊密和堆積性生長(zhǎng)變?yōu)榧忓N形、細(xì)胞伸長(zhǎng)和細(xì)胞之間接觸較不緊密的生長(zhǎng)。說(shuō)明細(xì)胞的分化類型發(fā)生了變化。Western blot進(jìn)一步檢測(cè)肺泡上皮細(xì)胞特異性蛋白標(biāo)志物表達(dá)水平，發(fā)現(xiàn)DDW可顯著上調(diào)水通道蛋白AQP5和T1a兩種Ⅰ型肺泡上皮特異性蛋白標(biāo)志物的表達(dá)，同時(shí)SPC1和SPC2兩種Ⅱ型上皮特異性蛋白標(biāo)志物的表達(dá)也顯著提高。上述細(xì)胞形態(tài)學(xué)表型和特異性標(biāo)志物水平的變化說(shuō)明DDW可誘導(dǎo)A549細(xì)胞由Ⅱ型肺泡上皮細(xì)胞向Ⅰ型肺泡上皮細(xì)胞轉(zhuǎn)化，從而導(dǎo)致A549細(xì)胞的分化程度提高，惡性增殖能力下降。

綜上所述，DDW可通過(guò)下調(diào)MAPK/ERK信號(hào)通路，阻滯A549細(xì)胞細(xì)胞周期，抑制細(xì)胞增殖；DDW還可以誘導(dǎo)A549細(xì)胞由Ⅱ型肺泡上皮向Ⅰ型肺泡上皮的轉(zhuǎn)化。這些研究結(jié)果部分闡明了DDW抑制肺癌細(xì)胞增殖的分子機(jī)制，為DDW用于肺癌的輔助治療提供了依據(jù)。

[1] Macrae RM. Isotopes and analogs of hydrogen--from fundamental investigations to practical applications[J]. Sci Prog, 2013,96(Pt3):237-293.

[2] Boros LG, D′Agostino DP, Katz HE, et al. Submolecular regulation of cell transformation by deuterium depleting water exchange reactions in the tricarboxylic acid substrate cycle[J]. Med Hypotheses, 2016,87:69-74.

[3] Somlyai G, Jancsó G, Jákli G, et al. Naturally occurring deuterium is essential for the normal growth rate of cells[J]. FEBS Letters, 1993,317(1-2):1-4.

[4] Ehleringer JR, Rundel PW, Nagy KA. Stable isotopes in physiological ecology and food web research[J]. Trends Ecol Evol, 1986,1(2):42-45.

[5] Gyongyi Z, Budan F, Szabo I, et al. Deuterium depleted water effects on survival of lung cancer patients and expression of Kras, Bcl2, and Myc genes in mouse lung[J]. Nutr Cancer, 2013,65(2):240-246.

[6] Cong FS, Zhang YR, Sheng HC, et al. Deuterium-depleted water inhibits human lung carcinoma cell growth by apoptosis[J]. Exp Ther Med, 2010,1(2):277-283.

[7] Krempels K, Somlyai I, Somlyai G. A retrospective evaluation of the effects of deuterium depleted water consumption on 4 patients with brain metastases from lung cancer[J]. Integr Cancer Ther, 2008,7(3):172-181.

[8] Roberts PJ, Der CJ. Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer[J]. Oncogene, 2007,26(22):3291-3310.

[9] Kondo H, Miyoshi K, Sakiyama S, et al. Differential regulation of gene expression of alveolar epithelial cell markers in human lung adenocarcinoma-derived A549 clones[J]. Stem Cells Int, 2015,2015:165867.

[10] Feillet C, van der Horst GT, Levi F, et al. Coupling between the circadian clock and cell cycle oscillators: implication for healthy cells and malignant growth[J]. Front Neurol, 2015,6:96.

[11] Yoon YK, Kim HP, Han SW, et al. KRAS mutant lung cancer cells are differentially responsive to MEK inhibitor due to AKT or STAT3 activation: implication for combinatorial approach[J]. Mol Carcinog, 2010,49(4):353-362.

[12] Chang F, Steelman LS, Shelton JG, et al. Regulation of cell cycle progression and apoptosis by the Ras/Raf/MEK/ERK pathway (Review)[J]. Int J Oncol, 2003,22(3):469-480.

[13] Foster KA, Oster CG, Mayer MM, et al. Characterization of the A549 cell line as a type Ⅱ pulmonary epithelial cell model for drug metabolism[J]. Exp Cell Res, 1998,243(2):359-366.

Effects of deuterium-depleted water on proliferation and differentiation of lung cancer A549 cells

QIBingxue,LIZiyi,CONGFengsong

(SchoolofLifeSciencesandBiotechnology,ShanghaiJiaotongUniversity,Shanghai200240,China)

Objective To study the effects of deuterium-depleted water (DDW) on human lung cancer cell line A549 and to explore the underlying molecular mechanisms. Methods A549 cells were randomly divided into two groups: the observation group and the control group, which were seperately cultured in the medium prepared with 50 ppm DDW and in the medium prepared with normal water for 30 d. The morphology of cells was observed under an inverted microscope. Cell growth was measured using the colorimetric cell counting kit-8 (CCK-8) reagent to compare the growth rate of cells between the two groups. The cells in the two groups were synchronized by serum starvation and then were separately stimulated with medium containing 10% serum of DDW and medium containing normal water. At 36 and 48 h after the stimulation, cells from each group were trypsinized, stained with propidium iodide (PI) and then were analyzed by fluorescence-activated cell sorting (FACS) to determine the percentages of cells distributed in G1, S and G2/M phases during cell cycle. Total proteins were extracted from cells cultured in normal medium or DDW medium. Western blotting was conducted to detect the expression levels of MEK1 and EGFR, phosphorylation of serine 217 and serine 298 on MEK1, and phosphorylation of tyrosine 1016 and tyrosine 1110. The expression levels of several alveolus-specific protein markers (type Ⅰ alveolus markers: AQP5 and T1a, type Ⅱ alveolus markers SPB, SPC1, SPC2 and CFTR) in cells of the two groups were also compared. Results In the control group, the cells were cuboidal and in close contact with accumulation of cells. In the observation group, the cells were spindle, and intercellular contact was loose with cell elongation. The OD of the observation group was lower than that of the control group (allP<0.05). After the serum starvation, cells were stimulated for 36 h, the percentage of cells in G1phase of the observation group was 71.75%, which was higher than that of the control group (57.01%), the percentage of cells in S phase was 22.43%, which was lower than that of the control group (35.88%), (allP<0.05). There was no significant difference in cell cycle ratio between the two groups at 48 h. No significant difference was found in the expression of MEK1, EGFR and the phosphorylation of EGFR on tyrosine 1016 and 1110 between these two groups. In the observation group, the phosphorylation of MEK1 on both serine 217 and serine 298 was significantly decreased (allP<0.01). The expression level of SPC2 in the observation group was significantly increased as compared with that of the control group (P<0.01).Conclusions Long-term cultivation of A549 lung cancer cells with DDW medium significantly inhibits the cell proliferation and differentiation, which is related to decreasing phosphorylation level of MEK1, arresting the G1/S phase transformation, inducing A549 cells from type Ⅱ alveolar epithelial cells to type I alveolar epithelial cells and improving the differentiation of A549 cells.

lung carcinoma; deuterium-depleted water; cell cycle; mitogen-activated extracellular signal-regulated kinase; cell differentiation

國(guó)家自然科學(xué)基金資助項(xiàng)目(81272478)；教育部重點(diǎn)實(shí)驗(yàn)室開(kāi)放課題(AF4150013)。

戚冰雪(1988-)，碩士研究生，主要研究方向?yàn)榈碗畬?duì)A549細(xì)胞的增殖抑制。E-mail: 647870061@qq.com

叢峰松(1970-)，副教授，主要研究方向?yàn)榭鼓[瘤藥物的研究與開(kāi)發(fā)。E-mail: fsongcong@sjtu.edu.cn

10.3969/j.issn.1002-266X.2017.06.001

R734.2

A

1002-266X(2017)06-0001-04

2016-11-14)