梁 碩,王志成,李艷博,,郭彩霞,龔守良,林承赫

(1.吉林大學公共衛(wèi)生學院衛(wèi)生部放射生物學重點實驗室,吉林長春 130021;2.首都醫(yī)科大學公共衛(wèi)生與家庭醫(yī)學學院,北京 100069;3.吉林大學第一醫(yī)院核醫(yī)學科,吉林長春 130021)

pshuttle-Egr-1-hSmac質(zhì)粒聯(lián)合X線照射對乳腺癌MDA-MB-435細胞增殖的抑制作用

梁 碩1,王志成1,李艷博1,2,郭彩霞2,龔守良1,林承赫3

(1.吉林大學公共衛(wèi)生學院衛(wèi)生部放射生物學重點實驗室,吉林長春 130021;2.首都醫(yī)科大學公共衛(wèi)生與家庭醫(yī)學學院,北京 100069;3.吉林大學第一醫(yī)院核醫(yī)學科,吉林長春 130021)

目的:構(gòu)建pshuttle-Egr-1-hSmac質(zhì)粒并轉(zhuǎn)染人乳腺癌MDA-MB-435細胞,觀察其抑制腫瘤細胞的輻射增敏作用。方法:轉(zhuǎn)染pshuttle-Egr-1-hSmac質(zhì)粒的MDA-MB-435細胞經(jīng)過2 Gy X線照射不同時間(4、8、12、24和48 h)和0.5～5.0 Gy X線照射后24 h收集細胞,采用RT-PCR和Western blotting法檢測Smac mRNA及其蛋白表達。將細胞分為對照組、pshuttle質(zhì)粒組、pshuttle-Egr-1-hSmac質(zhì)粒組、2 Gy組、pshuttle＋2.0 Gy組和pshuttle-Egr-1-hSmac＋2.0 Gy組,MTT法檢測各組細胞增殖;克隆形成實驗檢測細胞存活能力;AnnexinⅤ-FITC雙染法檢測細胞凋亡;PI單染法檢測細胞周期。結(jié)果:對照組和pshuttle質(zhì)粒組MDA-MB-435細胞中Smac mRNA無表達,而pshuttle-Egr-1-hSmac質(zhì)粒組MDA-MB-435細胞Smac m RNA表達水平隨時間延長逐漸升高,于24和48 h時表達水平最高;經(jīng)0.5～5.0 Gy X線照射后24 h MDA-MB-435細胞Smac m RNA表達水平隨照射劑量增加而逐漸增加,在2.0和5.0 Gy X線照射后Smac m RNA表達水平最高。pshuttle-Egr-1-hSmac質(zhì)粒組4、8、12和24 h后Smac蛋白表達水平逐漸升高,24 h后表達水平最高。經(jīng)過0、0.5、1.0、2.0和5.0 Gy X線照射后24 h Smac蛋白表達水平逐漸升高,尤其以5.0 Gy X線照射時表達水平最高。MTT法檢測時程效應,2.0 Gy、pshuttle＋2.0 Gy和pshuttle-Egr-1-hSmac＋2.0 Gy質(zhì)粒組24、48和72 h細胞A490值明顯低于對照組(P＜0.01);劑量效應,pshuttle-Egr-1-hSmac質(zhì)粒組1.0～5.0 Gy X線照射后, MDA-MB-435細胞A490值明顯低于0 Gy X線照射(P＜0.05或P＜0.01)。pshuttle-Egr-1-hSmac質(zhì)粒組細胞存活分數(shù)明顯低于對照組(P＜0.01)。pshuttle-Egr-1-hSmac＋2.0 Gy組細胞凋亡率明顯高于2.0 Gy組(P＜0.01), G0/G1期和S期細胞百分率明顯低于2.0 Gy照射組(P＜0.01),G2/M期細胞百分率明顯高于2.0 Gy組(P＜0.01)。結(jié)論:X線照射能增加pshuttle-Egr-1-hSmac質(zhì)粒轉(zhuǎn)染的MDA-MB-435細胞有效表達Smac m RNA及蛋白,能抑制細胞存活,且誘導G2/M期阻滯和凋亡增加;Smac基因聯(lián)合放射治療可明顯增加乳腺癌細胞的放射敏感性。

Smac基因;Egr-1啟動子;X射線;基因-放射治療;細胞凋亡

臨床放射治療(放療)在乳腺癌治療中占有重要的地位,但是輻射的副作用不可避免,如何在提高乳腺癌放療療效的同時減少副損傷是腫瘤治療的研究熱點[1-3]。輻射敏感啟動子Egr-1具有輻射誘導特性,即該啟動子在正常條件下不增強下游基因的表達,只有在輻射條件下,下游基因才能在Egr-1介導下表達增強,增強該基因的效應。因此Egr-1可以從時間和空間上調(diào)控下游基因的體內(nèi)表達,提高放療的敏感性,減輕毒副作用,實現(xiàn)腫瘤綜合治療的目的[4-5]。與Egr-1相連接的基因有很多種類,但是有關(guān)Smac基因的研究卻未見報道。本研究利用放療能夠殺傷腫瘤細胞和誘導Egr-1啟動子的轉(zhuǎn)錄作用以及Smac基因能夠促進腫瘤細胞凋亡的作用[6-7],將pshuttle-Egr-1-hSmac質(zhì)粒聯(lián)合X線照射作用于乳腺癌MDA-MB-435細胞,以降低輻射劑量并能夠減輕或避免正常組織損傷,達到抑制腫瘤生長和殺傷腫瘤細胞的目的,為腫瘤治療開辟新途徑。

1 材料與方法

1.1 細胞培養(yǎng)及轉(zhuǎn)染人乳腺癌細胞株MDA-MB-435為吉林大學衛(wèi)生部放射生物學重點實驗室保存,以含10%胎牛血清的DMEM培養(yǎng)液(美國Gibco公司)于37℃、5%CO2培養(yǎng)箱中常規(guī)培養(yǎng),待細胞80%～90%融合可傳代。取對數(shù)生長期細胞接種于6孔板,6×105個/孔。第2天待細胞達90%～95%,按照Lipofectamine 2000轉(zhuǎn)染試劑盒(美國Invitrogen公司)說明書轉(zhuǎn)染構(gòu)建正確的質(zhì)粒。

1.2 細胞照射采用國產(chǎn)X射線深部治療機進行照射,電壓200 k V,電流10 m A,濾板為0.5 mm Cu和1.0 mm Al。照射時靶皮距50 cm,劑量率0.287 Gy·min－1。

1.3 Smac m RNA和蛋白表達規(guī)律將經(jīng)過脂質(zhì)體轉(zhuǎn)染空載體pshuttle和pshuttle-Egr-1-hSmac質(zhì)粒24 h的MDA-MB-435細胞經(jīng)過2.0 Gy X線照射后不同時間(4、8、12、24和48 h)和不同劑量(0.5、1.0、2.0和5.0 Gy)照射后24 h收集細胞,采用RT-PCR方法和Western blotting法檢測Smac m RNA和蛋白表達規(guī)律,Smac引物序列上游引物:5′-gctctagaatggcggctctgaagagttggctgt-3′, 含XbaⅠ酶切位點;下游引物:5′-gcggatcctcaa tcctcacgcaggt-3′,含Bam HⅠ酶切位點。

1.4 細胞增殖時程及劑量效應實驗MDA-MB-435細胞分為對照組(control)、pshuttle質(zhì)粒組、pshuttle-Egr-1-hSmac質(zhì)粒組、2.0 Gy組、pshuttle＋2.0 Gy組和pshuttle-Egr-1-hSmac＋2.0 Gy組。MDA-MB-435細胞接種于96孔板, 1×104個/孔,每組6復孔。轉(zhuǎn)染質(zhì)粒24 h后,給予2.0 Gy照射,于照射后0、12、24、48和72 h加入MTT,每孔MTT(5 g·L－1)20μL,繼續(xù)培養(yǎng)4 h后棄上清,每孔加入150μL DMSO,待充分溶解后酶標儀測定490 nm波長處吸光度(A490)值。以A490值表示細胞增殖情況,A490值降低,細胞生長受抑制。

MDA-MB-435細胞以6×104個/孔接種于96孔細胞培養(yǎng)板,分別設對照組、pshuttle組和pshuttle-Egr-1-hSmac組,經(jīng)脂質(zhì)體轉(zhuǎn)染后24 h給予照射,照射劑量分別為0、0.5、1.0、2.0和5.0 Gy,X線照射后24 h加入MTT測量A490值。以A490值表示細胞增殖情況,A490值降低,細胞生長受抑制。

1.5 細胞克隆形成實驗對照細胞及轉(zhuǎn)染pshuttle和pshuttle-Egr-1-hSmac質(zhì)粒24 h后,按照預先設計的細胞密度接種到60 mm無菌塑料培養(yǎng)皿中,各組設3個平行樣,接種密度為100個/ 孔(0 Gy)、200個/孔(2 Gy)、600個/孔(4 Gy)、2 000個/孔(6 Gy)、5 000個/孔(8 Gy)和50 000個/孔(10 Gy)。加入5 m L培養(yǎng)液,繼續(xù)培養(yǎng)14 d,Giemsa染色,空氣中干燥,封片,計數(shù)克隆數(shù)。用未照射組的集落形成率(PE)進行校正,計算細胞存活分數(shù)(survival fraction,SF),計算導致細胞63%死亡所需劑量(D0)值。

1.6 細胞周期及凋亡檢測分組同1.4。MDAMB-435細胞接種于6孔板,6×105個/孔,細胞轉(zhuǎn)染24 h后給予2.0 Gy照射,24 h后胰酶消化收獲細胞,PBS洗2次,0.5 m L PBS重懸細胞,加入AnnexinⅤ/PI或PI避光染色1 h,立即上流式細胞儀進行檢測。采用CellQuest軟件收取細胞(每份樣品收取1×104個細胞),采用ModFit軟件分析,結(jié)果以細胞凋亡率及各周期細胞百分率表示。

1.7 統(tǒng)計學分析采用SPSS 17.0軟件進行統(tǒng)計分析。各組細胞A490值、細胞SF、凋亡率和各細胞周期細胞百分率以±s表示,均數(shù)兩兩比較采用t檢驗,多組均數(shù)比較采用完全隨機設計的單因素方差分析。

2 結(jié)果

2.1 X線照射后轉(zhuǎn)染pshuttle-Egr-1-hSmac質(zhì)粒的MDA-MB-435細胞中Smac mRNA表達的規(guī)律

2.0 Gy X線照射后,對照組和pshuttle質(zhì)粒組Smac mRNA在24 h時無表達,而pshuttle-Egr-1-hSmac質(zhì)粒組MDA-MB-435細胞中Smac m RNA表達水平隨時間延長逐漸增高,4、8、12、24和48 h時Smac m RNA表達相對值分別為0.55、0.52、0.81、1.63和1.59,于24和48 h時表達較高,具有一定的時程效應。見圖1。

重組質(zhì)粒轉(zhuǎn)染細胞后24 h進行照射,照射劑量為0.5、1.0、2.0和5.0 Gy,照射后24 h檢測Smac m RNA表達。對照組和pshuttle質(zhì)粒組MDA-MB-435細胞中Smac m RNA無表達。pshuttle-Egr-1-hSmac質(zhì)粒組MDA-MB-435細胞經(jīng)0.5、1.0、2.0和5.0 Gy照射后Smac m RNA表達相對值分別為0.31、0.84、1.33和1.29,其中2.0和5.0 Gy照射后Smac m RNA表達較為明顯,具有一定的劑量效應。見圖2。

圖1 2.0 Gy X線照射后不同時間MDA-MB-435細胞中Smac m RNA表達電泳圖Fig.1 Electrophoregram of Smac mRNA expressions in MDA-MB-435 cells at different time after 2.0 Gy X-ray irradiationLane 1:Pshuttle group;Lane 2:DL 2000 marker;Lane 3:Control group;Lane 4―8:4,8,12,24,and 48 h in pshuttle-Egr-1-hSmac＋2.0 Gy group.

圖2 不同劑量X線照射后24 h Smac m RNA表達電泳圖Fig.2 Electrophoregram of Smac m RNA expressions 24 h after different doses of X-ray irradiationLane 1:Control group;Lane 2:DL 2000 marker;Lane 3:Pshuttle group;Lane 4－7:5.0,2.0,10,and 0.5 Gy X-ray irradiation in pshuttle-Egr-1-hSmac plasmid group.

2.2 X線照射后轉(zhuǎn)染pshuttle-Egr-1-hSmac質(zhì)粒的MDA-MB-435細胞中Smac蛋白表達的規(guī)律轉(zhuǎn)染pshuttle-Egr-1-hSmac質(zhì)粒24 h后進行照射,采用Western blotting法檢測細胞中Smac蛋白表達。Smac的相對分子質(zhì)量為25 000,從圖3中可見有明顯的蛋白條帶,與Smac蛋白大小一致,而β-actin的相對分子質(zhì)量為42 000。MDA-MB-435細胞經(jīng)2.0 Gy X線照射后,對照組Smac蛋白無表達,pshuttle-Egr-1-hSmac質(zhì)粒組4、8、12和24 h后蛋白表達逐漸增加,灰度分析后蛋白表達相對值為0.28、0.31、0.44和0.62(圖3);不同劑量X線照射后,對照組無Smac蛋白表達,0.5、1.0、2.0和5.0 Gy照射組Smac蛋白表達相對值分別為0.31、0.34、0.68和0.86(圖4)。說明質(zhì)粒pshuttle-Egr-1-hSmac在X射線誘導下可有效表達Smac蛋白,并且以5 Gy照射后24 h表達最為明顯。

2.3 重組質(zhì)粒聯(lián)合X線照射對MDA-MB-435細胞生長抑制的時程及劑量效應時程效應結(jié)果:轉(zhuǎn)染pshuttle和pshuttle-Egr-1-hSmac質(zhì)粒后MDA-MB-435細胞生長有抑制傾向,但不明顯, 2.0 Gy、pshuttle＋2.0 Gy和pshuttle-Egr-1-hSmac＋2.0 Gy組細胞A490值明顯降低,24、48 和72 h時與對照組比較差異有統(tǒng)計學意義(P＜0.05或P＜0.01),其中pshuttle-Egr-1-hSmac＋2.0 Gy組細胞A490值降低最明顯,與pshuttle-Egr-1-hSmac組比較差異也有統(tǒng)計學意義(P＜0.05或P＜0.01)。劑量效應結(jié)果:MDA-MB-435細胞A490值隨照射劑量增加逐漸降低,對照組和pshuttle組降低不明顯;pshuttle-Egr-1-hSmac質(zhì)粒組MDA-MB-435細胞A490值明顯降低,1.0、2.0和5.0 Gy X線照射后,與0 Gy X線照射比較,細胞A490值明顯降低(P＜0.05或P＜0.01)。見表1和2。

圖3 2.0 Gy X線照射后不同時間Smac蛋白表達電泳圖Fig.3 Electrophoregram of Smac protein expressions at different time after 2.0 Gy X-ray irradiationLane 1―4:4,8,12,and 24 h after 2.0 Gy X-ray irradiation in pshuttle-Egr-1-hSmac＋2 Gy group;Lane 5:Control group.

圖4 不同劑量X線照射后24 h Smac蛋白表達電泳圖Fig.4 Electrophoregram of Smac protein expressions after different doses of X-ray irradiation for 24 hLane 1－4:0.5,1.0,2.0,and 5.0 Gy X-ray irradiation in pshuttle-Egr-1-hSmac plasmid group;Lane 5:Control group.

表1 2.0 Gy X線照射后不同時間各組細胞A490值Tab.1 A490values of cells after 2 Gy irradiation in various groups(n＝6±s)

表1 2.0 Gy X線照射后不同時間各組細胞A490值Tab.1 A490values of cells after 2 Gy irradiation in various groups(n＝6±s)

?P＜0.05,??P＜0.01 vs control group;△P＜0.05,△△P＜0.01 vs pshuttle-Egr1-hSmac group.

Group A490value (t/h) 0 12 24 48 72 Control 1 1 1 1 1 Pshuttle 0.97±0.04 0.97±0.05 0.95±0.05 0.98±0.04 0.91±0.08 Pshuttle-Egr-1-hSmac 0.95±0.08 0.99±0.04 1.02±0.04 0.97±0.03 0.95±0.02 2.0 Gy 0.91±0.01 0.89±0.05 0.89±0.05?0.86±0.08?0.89±0.02?Pshuttle＋2.0 Gy 0.92±0.09 0.95±0.05 0.86±0.03?0.93±0.08?0.87±0.06?Pshuttle-Egr-1-hSmac＋2.0 Gy 0.83±0.05?△0.73±0.02??△0.69±0.02??△△0.67±0.07??△△0.65±0.06??△△

表2 不同劑量X線照射后各組細胞A490值Tab.2 A490values of cells after different doses X-ray irradiation in various groups(n＝6,±s)

表2 不同劑量X線照射后各組細胞A490值Tab.2 A490values of cells after different doses X-ray irradiation in various groups(n＝6,±s)

?P＜0.05,??P＜0.01 vs control group.

Group A490value (D/Gy)0 0.5 1.0 2.0 5.0 Control 1 1 1 1 1 Pshuttle 1.01±0.04 1.02±0.05 1.02±0.03 1.01±0.02 0.99±0.07 Pshuttle-Egr-1-hSmac 1.01±0.02 0.94±0.05 0.91±0.02?0.87±0.02?0.71±0.02??

2.4 各組細胞劑量存活曲線與對照組比較, pshuttle組細胞SF未見明顯變化,而pshuttle-Egr-1-hSmac質(zhì)粒組細胞SF明顯降低(P＜0.01,見表3)。將各組細胞的SF進行直線相關(guān)與回歸分析得出如下方程:Y＝－9.9316X＋102.45, R2＝0.9661(對照組);Y＝－9.6487X＋100.62,R2＝0.9559(pshuttle組);Y＝－9.2199X＋80.614,R2＝0.9126(pshuttle-Egr-1-hSmac組)。進而計算得出各組細胞的D0值為3.31、3.29和2.70,對照組與pshuttle質(zhì)粒組細胞D0值相近,而pshuttle-Egr-1-hSmac質(zhì)粒組細胞D0值明顯降低,說明該組細胞放射敏感性高。

表3 各組MDA-MB-435細胞SFTab.3 SF of MDA-MB-435 cells in various groups(n＝3,±s,η/%)

表3 各組MDA-MB-435細胞SFTab.3 SF of MDA-MB-435 cells in various groups(n＝3,±s,η/%)

?P＜0.01 compared with control group.

Group SF (D/Gy)0 2 4 6 8 10 Control 99.00±1.11 85.20±3.32 70.30±4.07 40.20±1.88 12.30±1.44 9.76±0.89 Pshuttle 95.10±2.32 84.90±5.98 72.30±2.55 39.00±5.14 13.10±1.61 9.78±1.11 Pshuttle-Egr-1-hSmac 90.20±1.29?66.10±3.15?30.20±1.50?11.10±0.36?4.02±0.47?0.61±0.08?

2.5 重組質(zhì)粒聯(lián)合X線照射作用下MDA-MB-435細胞凋亡率MDA-MB-435細胞轉(zhuǎn)染pshuttle-Egr-1-hSmac質(zhì)粒后,細胞凋亡率未見明顯增加;與對照組比較,2.0 Gy、pshuttle＋2.0 Gy和pshuttle-Egr-1-hSmac＋2.0 Gy組細胞凋亡率明顯升高(P＜0.05或P＜0.01),其中以pshuttle-Egr-1-hSmac＋2.0 Gy組升高最為明顯;與2.0 Gy組比較,pshuttle-Egr-1-hSmac＋2.0 Gy組細胞凋亡率明顯升高(P＜0.01)。見表4。

表4 各組MDA-MB-435細胞凋亡率Tab.4 Apoptotic rates of MDA-MB-435 cells in various groups(n＝4,±s,η/%)

表4 各組MDA-MB-435細胞凋亡率Tab.4 Apoptotic rates of MDA-MB-435 cells in various groups(n＝4,±s,η/%)

?P＜0.05,??P＜0.01 vs control group;△P＜0.01 vs 2.0 Gy group.

Group Apoptotic rate Control 3.71±0.69 Pshuttle 4.13±0.88 Pshuttle-Egr-1-hSmac 4.03±0.45 2.0 Gy 5.12±1.31?Pshuttle＋2.0 Gy 5.23±1.09?Pshuttle-Egr-1-hSmac＋2.0 Gy 29.12±4.15??△

2.6 重組質(zhì)粒聯(lián)合X線照射作用下MDA-MB-435細胞周期的細胞百分率對照組、pshuttle組和pshuttle-Egr-1-hSmac組各期MDA-MB-435細胞百分率基本一致;2.0 Gy X線照射后,2.0 Gy組、pshuttle＋2.0 Gy組和pshuttle-Egr-1-hSmac＋2.0 Gy組G0/G1和S期細胞百分率明顯降低(P＜0.01),而G2/M期細胞百分率明顯升高(P＜0.01),其中pshuttle-Egr-1-hSmac＋2.0 Gy組細胞百分率與對照組、2.0 Gy組和pshuttle-Egr-1-hSmac組比較差異均有統(tǒng)計學意義(P＜0.01)。見表5。

3 討論

放療是乳腺癌治療的一個重要手段,但是放療所致鄰近部位的放射損傷或者患者的輻射耐受嚴重影響和限制其療效和廣泛應用,如何增強乳腺癌細胞對射線的敏感性對提高乳腺癌預后非常重要[8-9]。輻射敏感啟動子Egr-1具有輻射誘導特性,該啟動子在正常條件下不增強下游基因的表達,只有在照射條件下,下游基因在Egr-1介導下表達增強,從而增強該基因的效應[10-11]。Egr-1可以從時間和空間上調(diào)控下游基因的體內(nèi)表達,提高放療的敏感性。Smac是2000年發(fā)現(xiàn)的一種存在于線粒體并且調(diào)節(jié)細胞凋亡的蛋白質(zhì),主要通過抑制凋亡抑制蛋白(inhibitor of apoptosis proteins,IAPs)的活性而發(fā)揮促凋亡作用,是一種線粒體依賴性凋亡途徑[12-15]。

表5 各組MDA-MB-435細胞周期細胞百分率Tab.5 Percentages of MDA-MB-435 cells at each cell cycle in various groups(n＝4,±s,η/%)

表5 各組MDA-MB-435細胞周期細胞百分率Tab.5 Percentages of MDA-MB-435 cells at each cell cycle in various groups(n＝4,±s,η/%)

?P＜0.05,??P＜0.01 vs control group;△P＜0.05,△△P＜0.01 vs pshuttle-Egr-1-hSmac group;＃P＜0.05,＃＃P＜0.01 vs 2.0 Gy group.

Group Percentage of MDA-MB-435 cells G0/G1S G2/M Control 61.28±1.64 31.56±1.11 8.16±0.64 Pshuttle 62.95±1.8 26.68±1.59 10.37±0.85 Pshuttle-Egr-1-hSmac 60.82±1.22 31.93±1.01 8.25±2.19 2.0 Gy 48.42±1.56?28.30±1.74?23.28±1.43??Pshuttle＋2.0 Gy 49.78±1.78?24.83±2.67?30.39±2.26??Pshuttle-Egr-1-hSmac＋2.0 Gy 36.92±1.83??△△＃22.18±0.85?△＃40.9±1.43??△＃

本研究利用已構(gòu)建成功的pshuttle-Egr-1-hSmac質(zhì)粒轉(zhuǎn)染人乳腺癌MDA-MB-435細胞,結(jié)果顯示:2.0 Gy X線照射后24 h,對照組和pshuttle質(zhì)粒組均無Smac m RNA表達,而轉(zhuǎn)染pshuttle-Egr-1-hSmac質(zhì)粒的細胞經(jīng)過2.0 Gy照射后,Smac mRNA表達水平隨著時間的延長逐漸升高,于照射后24和48 h表達較高,具有一定的時程效應關(guān)系;pshuttle-Egr-1-hSmac質(zhì)粒組轉(zhuǎn)染后進行0.5～5.0 Gy X線照射,細胞中Smac mRNA表達具有一定的量效關(guān)系,在2.0和5.0 Gy照射后表達較為明顯;蛋白檢測結(jié)果顯示: pshuttle組MDA-MB-435細胞中無Smac蛋白表達,經(jīng)0.5、1.0、2.0和5.0 Gy照射后表達逐漸增多,5.0 Gy照射時其蛋白表達最為明顯, 2.0 Gy照射后4 h開始即有表達且逐漸增多,照射后24 h時達到最高。本研究結(jié)果提示:在X線照射后,從Smac m RNA和蛋白水平上看,Egr-1啟動子發(fā)揮了輻射誘導增強的作用,具有一定的時程效應和劑量效應關(guān)系,而空載體和正常對照則無表達。

本研究中MTT檢測結(jié)果顯示:MDA-MB-435細胞在轉(zhuǎn)染pshuttle和pshuttle-Egr-1-hSmac質(zhì)粒后0～72 h,細胞增殖能力稍有降低,但不明顯;而各組細胞經(jīng)過2.0 Gy X射線照射后,細胞增殖能力明顯降低,尤其以轉(zhuǎn)染了pshuttle-Egr-1-hSmac質(zhì)粒的細胞增殖能力降低的更明顯;0～5.0 Gy X射線照射后,對照組MDA-MB-435細胞增殖能力降低,但差異不明顯;轉(zhuǎn)染pshuttle質(zhì)粒后進行相應劑量照射,轉(zhuǎn)染了pshuttle-Egr-1-hSmac質(zhì)粒則明顯抑制細胞增殖。上述結(jié)果表明:轉(zhuǎn)染重組質(zhì)粒對MDA-MB-435細胞生長影響不明顯,而X射線照射能抑制MDA-MB-435細胞生長,在轉(zhuǎn)染后進行照射則可加強其抑制效果,可能與輻射誘導Smac表達有關(guān)。本研究結(jié)果顯示:轉(zhuǎn)染空載體pshuttle后,MDA-MB-435細胞SF未見明顯改變,即轉(zhuǎn)染空載體未對細胞產(chǎn)生明顯影響,而轉(zhuǎn)染pshuttle-Egr-1-hSmac質(zhì)粒的細胞SF明顯下降,說明MDA-MB-435細胞存活能力明顯降低,輻射對其具有明顯影響。本研究結(jié)果顯示:對照組和pshuttle質(zhì)粒組D0值基本相同,說明放射敏感性無明顯差別;而pshuttle-Egr-1-hSmac質(zhì)粒組的D0值降低,說明細胞放射敏感性增高,提示轉(zhuǎn)染pshuttle-Egr-1-hSmac質(zhì)粒能增強MDA-MB-435細胞的放射敏感性;電離輻射可以抑制乳腺癌細胞的生長,轉(zhuǎn)染pshttle-Egr-1-hSmac質(zhì)粒后進行相應電離輻射細胞生長抑制更明顯,可能與電離輻射誘導Smac表達有關(guān)。

本研究中凋亡檢測結(jié)果顯示:MDA-MB-435細胞轉(zhuǎn)染pshuttle-Egr-1-hSmac質(zhì)粒后,凋亡率未增加;2.0 Gy照射后,與對照組比較,pshuttle-Egr-1-hSmac組細胞凋亡率明顯升高,與其他組比較差異均有統(tǒng)計學意義,說明基因聯(lián)合放療對乳腺癌MDA-MB-435細胞的促凋亡作用具有良好的效果,能有效地誘導細胞凋亡、殺傷細胞。Smac基因是線粒體內(nèi)釋放的凋亡促進蛋白,而電離輻射也可以誘導細胞凋亡,二者的聯(lián)合應用勢必增加細胞凋亡率。本研究結(jié)果顯示:轉(zhuǎn)染pshuttle和pshuttle-Egr-1-hSmac后MDA-MB-435細胞周期無明顯變化,2.0 Gy、pshuttle＋2.0 Gy和pshuttle-Egr-1-hSmac＋2.0 Gy組MDA-MB-435細胞G0/G1和S期細胞百分率明顯下降,G2/M期細胞百分率明顯升高,其中pshuttle-Egr-1-hSmac ＋2.0 Gy組變化最為明顯,導致MDA-MB-435細胞G2/M期阻滯。上述結(jié)果可能與電離輻射的作用有密切關(guān)聯(lián),也是輻射誘導細胞凋亡的主要機制之一。

綜上所述,pshuttle-Egr-1-hSmac聯(lián)合X線照射能有效地抑制MDA-MB-435細胞的生長,提高細胞放射敏感性,影響細胞周期進程,導致MDA-MB-435細胞發(fā)生G2/M期阻滯,促進細胞凋亡,從而達到更好的殺傷腫瘤的作用。本研究為提高基因-放療效果開辟了新途徑,為基因-放療的臨床應用提供了實驗依據(jù)。

[1]Cammarota F,Giugliano FM,Iadanza L,et al. Hypofractionated breast cancer radiotherapy.Helical tomotherapy in supine position or classic 3D-conformal radiotherapy in prone position:Which is better?[J].Anticancer Res,2014,34(3):1233-1238.

[2]Auer J,Keller U,Schmidt M,et al.Individual radiosensitivity in a breast cancer collective is changed with the patients’age[J].Radiol Oncol,2014,48(1):80-86.

[3]Belkacemi Y,Khodari W,Grelier N,et al.Breast cancer radiotherapy:current changes[J].Rev Prat,2013, 63(10):1402-1403.

[4]Woo SM,Min KJ,Kim S,et al.Silibinin induces apoptosis of HT29 colon carcinoma cells through early growth response-1(EGR-1)-mediated non-steroidal antiinflammatory drug-activated gene-1(NAG-1) up-regulation[J].Chem Biol Interact,2014,211(4):36-43.

[5]Li YB,Guo CX,Wang ZC,et al.Radiosensitization of breast cancer cells by TRAIL-endostatin-targeting gene therapy[J].Neoplasma,2013,60(6):613-619.

[6]Monteiro JP,Oliveira PJ,Jurado AS.Mitochondrial membrane lipid remodeling in pathophysiology:a new target for diet and therapeutic interventions[J].Prog Lipid Res, 2013,52(4):513-528.

[7]Wang K,Lin B.Inhibitor of apoptosis proteins(IAPs)as regulatory factors of hepatic apoptosis[J].Cell Signal,2013, 25(10):1970-1980.

[8]Badakhshi H,Gruen A,Sehouli J,et al.The impact of patient compliance with adjuvant radiotherapy:a comprehensive cohort study[J].Cancer Med,2013,2(5): 712-717.

[9]Blitzblau RC,Horton JK.Radiotherapy after mastectomy[J].Surg Oncol Clin N Am,2013,22(3): 563-577.

[10]Hu Y,Ouyang W,Wu F,et al.Enhanced radiosensitivity of SW480 cells via TRAIL up-regulation mediated by Egr-1 promoter[J].Oncol Rep,2009,22(4):765-771.

[11]Zhou Y,Song X,Jia R,et al.Radiation-inducible human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)gene therapy:a novel treatment for radioresistant uveal melanoma[J].Pigment Cell Melanoma Res,2010, 23(5):661-674.

[12]Moon D,McCormack D,McDonald D,et al.Pterostilbene induces mitochondrially derived apoptosis in breast cancer cells in vitro[J].J Surg Res,2013,180(2):8-15.

[13]Hsiao WT,Tsai MD,Jow GM,et al.Involvement of Smac,p53,and caspase pathways in induction of apoptosis by gossypol in human retinoblastoma cells[J].Mol Vis, 2012,18(20):2033-2042.

[14]Allensworth JL,Sauer SJ,Lyerly HK,et al.Smac mimetic Birinapant induces apoptosis and enhances TRAIL potency in inflammatory breast cancer cells in an IAP-dependent and TNF-α-independent mechanism[J].Breast Cancer Res Treat, 2013,137(2):359-371.

[15]Yang D,Zhao Y,Li AY,et al.Smac-mimetic compound SM-164 induces radiosensitization in breast cancer cells through activation of caspases and induction of apoptosis[J].Breast Cancer Res Treat,2012,133(1):189-199.

Inhibitory effect of pshuttle-Egr-1-hSmac plasmid combined with X-ray irradiation on proliferation of breast cancer MDA-MB-435 cells

LIANG Shuo1,WANG Zhi-cheng1,LI Yan-bo1,2,GUO Cai-xia2,GONG Shou-liang1,LIN Cheng-he3
(1.Key Laboratory of Radiobiology,Ministry of Health,School of Public Health,Changchun 130021, China;2.School of Public Health and Family Medicine,Capital Medical University,Beijing 100069, China;3.Department of Nuclear Medicine,First Hospital,Jilin University,Changchun 130021,China)

ObjectiveTo construct the pshuttle-Egr-1-hSmac plasmid and transfect human breast cancer MDA-MB-435 cells,and to observe its radiotherapy enhancing effect on tumor cells.MethodsThe empty vector pshuttle and pshuttle-Egr-1-hSmac plasmid were transfected into MDA-MB-435 cells by liposomal.At different time(4,8,12,24 and 48 h)after irradiation with 2.0 Gy X-ray and 24 h after irradiation with 0.5―5.0 Gy,the total RNA and protein were collected and extracted from these cells to analyze the Smac m RNA and protein expression levels with RT-PCR and Western blotting methods.The cells were divided into control,pshuttle, pshuttle-Egr-1-hSmac,2.0 Gy irradiation group,pshuttle＋2.0 Gy irradiation and pshuttle-Egr-1-hSmac＋2.0 Gy irradiation groups.MTT method was used to evaluate cell proliferation,and the cell survival ability was measured with clone formation assay;AnnexinⅤ/PI double staining and PI single staining were used to examine the apoptosis and cell cycle of MDA-MB-435 cells.ResultsThere was no Smac m RNA expression in MDA-MB-435 cells in control and pshuttle groups,but the Smac mRNA expression levels in MDA-MB-435 cells in pshuttle-Egr-1-hSmac plasmid group were gradually increased with the time prolongation,and reached the maximum at 24 and 48 h;the Smac m RNA expression levels in MDA-MB-435 cells were increased gradually 24 h after irradiation of 0.5―5.0 Gy X-ray with the increasing of irradiation doses,and reached the maximum after 2.0 and 5.0 Gy irradiation.The Smac protein expression levels in pshuttle-Egr-1-hSmac plasmid group were increased gradually with the time prolongation,and reached the maximum at 24 h.The Smac protein expression lervels were increased 24 h afer irradiation of 0,0.5,1.0,2.0 and 5.0 Gy X-ray,especially in 5.0 Gy group.The MTT results showed that the A490values in 2.0 Gy,pshuttle＋2.0 Gy and pshuttle-Egr-1-hSmac groups 24, 48,and 72 h after irradiation were lower than those in control group(P＜0.01);the A490values of MDA-MB-435 cells in pshuttle-Egr-1-hSmac group after 1.0－5.0 Gy X-ray irradiation were lower than those in 0 Gy group(P＜0.05 or P＜0.01);the survival fraction(SF)in pshuttle-Egr-1-hSmac group was lower than those in control group (P＜0.01).The percentages of the cells at G0/G1and S phase in pshuttle-Egr-1-hSmac group were lower than those in 2.0 Gy group(P＜0.01),the percentage of the cells at G2/M phase was higher than that in 2.0 Gy group(P＜0.01);the apoptotic rate of the cells in pshuttle-Egr-1-hSmac group was higher than that in 2.0 Gy group(P＜0.01).ConclusionX-ray irradiation can significantly increase the Smac mRNA and protein expression levels in MDA-MB-435 cells transfected with pshuttle-Egr-1-hSmac plasmid,inhibit the cell survival rate,and induce G2/M arrest and apoptotic increasing;Smac gene combined with radiotherapy could significantly increase the radiosensitivity of breast cancer cells.

Smac gene;Egr-1 promoter;X-ray;gene-radiotherapy;apoptosis

R737.9

A

2014-01-10

國家自然科學基金資助課題(30870747);吉林大學基本科研項目資助課題(2012)

梁 碩(1973－),男,吉林省長春市人,講師,醫(yī)學博士,主要從事腫瘤基因-放射治療方面的研究。

林承赫(Tel:0431-88782717,E-mail:linchh1967＠163.com)