時(shí)迎娣,張迎秋,倪揚(yáng)笑,史國利,楊懷義
1 中國科學(xué)院微生物研究所,北京 100101
2 中國科學(xué)院研究生院,北京 100049
3 遼寧師范大學(xué),遼寧 大連 116029
隨著人們飲食結(jié)構(gòu)和生活方式的改變,我國的前列腺癌發(fā)病率和死亡率呈現(xiàn)逐年上升趨勢(shì)[1-2]。針對(duì)早期前列腺癌細(xì)胞具有雄激素依賴性的特征,前期治療前列腺癌最有效的手段,是通過雄激素阻斷療法進(jìn)行治療,然而在治療期間,前列癌細(xì)胞往往會(huì)由雄激素依賴性轉(zhuǎn)變?yōu)樾奂に胤且蕾囆訹3-5]。目前,還沒有有效的手段或藥物治療雄激素非依賴性前列腺癌。因此,雄激素非依賴性前列腺癌的治療成為前列腺癌治療的難點(diǎn)和重點(diǎn)。
PC3M細(xì)胞是前列腺癌高轉(zhuǎn)移細(xì)胞系,是一種研究抑制前列腺癌雄激素非依賴性癌細(xì)胞生長的常用細(xì)胞[6-10]。近年來,人們研究了DIM[6]、金絲桃素[7]、羥基喜樹堿[8]、苦參堿[9]、茶多酚[10]等對(duì)該細(xì)胞的抑制作用,為雄激素非依賴性前列腺癌的治療提供了新的線索。
11-脫氧輪枝菌素A (又稱11′-脫氧沃替西林A,11'-deoxyverticillin A,C42),是一種從冬蟲夏草共生菌中分離得到的多硫代二氧基哌嗪(Epipolythiodioxopiperazines,ETPs) 族結(jié)構(gòu)化合物[11],其對(duì)結(jié)腸癌細(xì)胞有誘導(dǎo)細(xì)胞凋亡的作用[12]。為探討其對(duì)前列腺癌雄激素非依賴性癌細(xì)胞生長的抑制作用,我們研究了其對(duì)PC3M細(xì)胞凋亡的影響。結(jié)果表明,C42具有誘導(dǎo) caspase依賴性細(xì)胞凋亡,抑制PC3M細(xì)胞生長的作用。這為進(jìn)一步研究利用 C42類化合物作為雄激素非依賴性前列腺癌的可能臨床藥物提供了一定的實(shí)驗(yàn)和理論參考。
雄激素非依賴性前列腺癌高轉(zhuǎn)移細(xì)胞系PC3M細(xì)胞為本實(shí)驗(yàn)室保存。
MEM、無酚紅DMEM培養(yǎng)基、胎牛血清購自Gibco公司;預(yù)染蛋白Marker購自Fermentas公司;β-Actin抗體購自北京中杉金橋公司,PARP抗體購自Sigma-Aldrich公司;ECL發(fā)光液購自GE公司;Caspase-Glo 3/7試劑盒、MTS試劑盒(CellTiter 96? Aqueous Non-Radioactive Cell Proliferation Assay) 購自Promega公司;Annexin V-FITC試劑盒購自BD公司。11'-Deoxyverticillin A (C42) 由中國科學(xué)院微生物研究所車永勝研究員提供。Sunrise酶標(biāo)儀為TECAN公司生產(chǎn);生物發(fā)光檢測(cè)儀為Promega公司生產(chǎn);流式細(xì)胞儀為BD公司生產(chǎn)。
PC3M細(xì)胞于完全MEM培養(yǎng)基 (10%胎牛血清、100 g/L鏈霉素和100 U/mL的青霉素) 中培養(yǎng),條件為37 ℃、5% CO2。
將5 000~10 000個(gè)細(xì)胞分至96孔板中,每孔加入100 μL完全MEM培養(yǎng)基 (含10%胎牛血清、100 g/L鏈霉素及100 U/mL的青霉素)。過夜后換為新鮮的無酚紅DMEM培養(yǎng)基 (含10%胎牛血清),并加入不同濃度的C42。加入20 μL MTS/PMS (MTS∶PMS=20∶1),37 ℃、5% CO2條件下持續(xù)培養(yǎng),4 h內(nèi)檢測(cè),酶標(biāo)儀測(cè)定490 nm處的OD值,每樣品設(shè)3次以上重復(fù)。
細(xì)胞經(jīng)不同濃度、不同時(shí)間C42處理后,PBS漂洗并加入細(xì)胞裂解液 (150 mmol/L NaCl,50 mmol/L Tris-HCl,2 mmol/L EDTA,0.5% TritonX-100,0.5%脫氧膽酸鈉和 1×蛋白酶抑制劑),收集裂解產(chǎn)物,冰上放置30 min,13 000 r/min離心15 min后收集上清。SDS-PAGE (8%) 電泳,濕轉(zhuǎn)法將蛋白轉(zhuǎn)移至PVDF膜。5%的脫脂牛奶封閉 2 h。孵育特異性抗體,anti-PARP為1∶1 000,anti-β-actin為1∶4 000,二抗分別為辣根過氧化物酶標(biāo)記的抗兔 IgG 1∶4 000或抗小鼠IgG 1∶2 000,加ECL發(fā)光液,X-ray自顯影后采用圖像系統(tǒng)軟件Quantity One分析,分析目的蛋白的表達(dá)差異。
將5 000~10 000個(gè)細(xì)胞分至96孔板中,每孔加入100 μL完全MEM培養(yǎng)基 (含10%胎牛血清、100 g/L鏈霉素及100 U/mL的青霉素)。過夜后換為新鮮的DMEM培養(yǎng)基 (含10%胎牛血清),并加入不同濃度的 C42,每孔100 μL。37 ℃、5% CO2條件下持續(xù)培養(yǎng)6 h后,于室溫下,加入100 μL Caspase-Glo 3/7試劑,300~500 r/min搖動(dòng)30 min,生物發(fā)光儀檢測(cè)熒光。每樣品設(shè)3次以上重復(fù)。
胰酶消化經(jīng)不同濃度C42處理的細(xì)胞,離心收集細(xì)胞,PBS洗滌 2次,加入 1×結(jié)合緩沖液(0.1 mol/L Hepes (pH 7.4),1.4 mol/L NaCl,25 mmol/L CaCl2) 重懸細(xì)胞,使其密度為1×106個(gè)細(xì)胞/mL。取出 1×105個(gè)細(xì)胞加入流式專用管中,并加入5 μL Annexin V-FITC,室溫下避光孵育15 min,上機(jī)前1 min,再加入1.5 μL PI,并補(bǔ)加400 μL 1×結(jié)合緩沖液,混勻細(xì)胞后,流式細(xì)胞儀分析。
通過MTS/PMS檢測(cè)C42處理對(duì)PC3M細(xì)胞增殖的影響。結(jié)果表明,使用濃度分別為0.25 μmol/L、0.5 μmol/L的C42處理PC3M細(xì)胞12 h后,其相對(duì)細(xì)胞增殖抑制率分別為11% (P<0.05) 及44% (P<0.01) (圖1A)。用濃度為0.25 μmol/L C42處理PC3M細(xì)胞,發(fā)現(xiàn)隨著時(shí)間的延長,PC3M細(xì)胞相對(duì)細(xì)胞增殖抑制率顯著增加 (圖1B)。如圖1B所示,PC3M細(xì)胞相對(duì)細(xì)胞增殖抑制率從無顯著變化 (6 h) 增加至 36% (24 h),其中12 h組與6 h組相比,24 h組與12 h組相比均具有顯著性差異 (P<0.01)。以上說明,C42具有抑制PC3M細(xì)胞生長的作用,并且對(duì)細(xì)胞生長的抑制作用同時(shí)間和濃度具有相關(guān)性。
圖1 C42誘導(dǎo)對(duì)PC3M細(xì)胞生長的影響Fig. 1 C42 inhibits the cell proliferation of prostate cancer cells. (A) Cell viability of PC3M cells treated with different concentrations of C42 for 12 h. (B) Cell viability of PC3M cells treated with C42 (0.25 μmol/L) for different time. The data is represented as from three independent experiments. *P<0.05; **P<0.01.
細(xì)胞凋亡是細(xì)胞死亡的一條重要途徑。為驗(yàn)證C42處理是否通過誘導(dǎo)PC3M細(xì)胞凋亡的發(fā)生,從而降低了其細(xì)胞增殖能力。我們利用細(xì)胞凋亡發(fā)生時(shí)膜內(nèi)外翻的磷脂酰絲氨酸可與Annexin V特異性結(jié)合,及PI能穿透不完整細(xì)胞膜使凋亡晚期細(xì)胞和壞死細(xì)胞的細(xì)胞核著色的特征,通過Annexin V/PI雙染,利用流式細(xì)胞術(shù)對(duì)其進(jìn)行了分析。結(jié)果表明,經(jīng)C42處理6 h后,細(xì)胞凋亡顯著增強(qiáng)。0.25 μmol/L 和 0.5 μmol/L C42處理后,其早期凋亡細(xì)胞增加數(shù)量與對(duì)照相比分別增加了1.1倍和2.6倍,均達(dá)到了統(tǒng)計(jì)學(xué)上的顯著差異 (圖2A,2B)。而晚期凋亡細(xì)胞和/或壞死細(xì)胞的增加數(shù)量,僅0.5 μmol/L的處理達(dá)到了顯著性差異 (圖2A,2C)。
圖2 C42對(duì)PC3M細(xì)胞凋亡的流式細(xì)胞分析Fig. 2 The relationship between C42 and cell apoptosis of PC3M cells. (A) After treated with different concentrations of C42 for 6 h, PC3M cells were collected, incubated with FITC-Annexin V/PI and subjected to flow cytometry. (B) Quantities of early apoptotic cells: PI negative and Annexin V positive cells in all analyzed PC3M cells. (C) Quantities of end stage apoptotic and/ or necrotic cells: PI positive cells in all analyzed PC3M cells. The data is represented as from three independent experiments. *P<0.05; **P<0.01.
圖3 C42對(duì)caspase-3/7的激活Fig. 3 Relationship between C42 and the activity of caspase-3/7 in PC3M cells. PC3M cells were pretreated with C42 for 6 h, then, cells were subjected to caspase-Glo 3/7 assay. The data represented is based on three independent experiments. **P<0.01.
Caspase-3/7在細(xì)胞發(fā)生凋亡過程中起到至關(guān)重要的作用,其酶活性隨著細(xì)胞凋亡發(fā)生程度的增加而增加。為驗(yàn)證C42處理PC3M細(xì)胞后誘導(dǎo)的細(xì)胞凋亡,是否具有caspase依賴性。我們用caspase-Glo 3/7試劑盒對(duì)caspase-3/7的活性進(jìn)行檢測(cè)。如圖 3所示,隨著 C42處理濃度的增加,caspase-3/7切割的底物所發(fā)的熒光強(qiáng)度不斷增加,當(dāng)C42濃度分別為0.1、0.25、0.5 μmol/L時(shí),其熒光強(qiáng)度與對(duì)照組相比均具有極顯著的差異,分別是對(duì)照組的2倍、5倍和6.5倍。說明C42能夠增強(qiáng)PC3M細(xì)胞的caspase-3/7的活性,誘導(dǎo)caspase依賴性細(xì)胞凋亡,且具有一定的濃度依賴性。
PARP的切割是 caspase依賴性細(xì)胞凋亡的一種特異性標(biāo)志,當(dāng)caspase依賴性細(xì)胞凋亡發(fā)生時(shí),116 kDa的PARP會(huì)被caspase特異性切割為89 kDa和24 kDa[13-14]。因此為進(jìn)一步闡明C42誘導(dǎo)的細(xì)胞凋亡為 caspase依賴的凋亡,我們?cè)诜治隽薱aspase-3/7活性的基礎(chǔ)上,對(duì)PARP的切割情況進(jìn)行了分析。結(jié)果發(fā)現(xiàn),當(dāng)分別用濃度為0.1、0.25、0.5 μmol/L的C42處理PC3M細(xì)胞12 h后,隨著C42濃度的增加,PARP蛋白的被剪切程度增強(qiáng) (圖 4A,4B)。當(dāng)用 0.25 μmol/L的C42處理細(xì)胞0、3、6、12 h時(shí),PARP的剪切量隨著時(shí)間的延長而不斷增加 (圖4C,4D)。PARP的剪切對(duì)C42具有一定的濃度依賴性和時(shí)間依賴性。這進(jìn)一步說明,C42對(duì)PC3M細(xì)胞誘導(dǎo)的細(xì)胞凋亡為caspase依賴的細(xì)胞凋亡。
圖4 C42對(duì)PC3M細(xì)胞PARP剪切的影響Fig. 4 Relationship between C42 and the expression levels of cleaved PARP in PC3M cells. (A) Western blotting analysis of the PARP expression levels in PC3M cells. PC3M cells were treated with different concentrations of C42 for 12 h, then cells were lysed and subjected to western blotting with antibody to PARP or β-actin. (B) Quantification of the cleaved PARP (cPARP) expression levels for A. The cPARP/A (%) and cPARP/PARP (%) in PC3M cells treated with 0.5 μmol/L C42 were defined as 100 respectively. The results were based on three independent experiments. (C) Western blotting analysis of the cPARP expression levels in PC3M cells. Treated with 0.25 μmol/L C42 for 0 h, 3 h, 6 h, and 12 h, PC3M cells were then lysed and subjected to Western blotting with the antibody to PARP or β-actin. (D) Quantification of the cPARP expression levels for C. The cPARP/A (%) and cPARP/PARP (%) in PC3M cells treated with C42 for 12 h were defined as 100 respectively. The results were based on three independent experiments. cPARP/A (%): Quantitation of the cPARP (89 kDa) expression levels normalized against β-actin. cPARP/PARP (%): Quantitation of the cPARP (89 kDa) expression levels normalized against the full-length PARP (116 kDa).
ETPs族化合物是來源于真菌的活性化合物,具有免疫抑制、抗炎癥反應(yīng)和抗菌、寄生蟲、病毒等廣泛的生物活性作用[15],是潛在的腫瘤治療藥物,其家族成員膠黏毒素、來普生苷均被證實(shí)對(duì)癌細(xì)胞產(chǎn)生毒性[16-18]。我們研究發(fā)現(xiàn),從冬蟲夏草共生菌中分離到的此類化合物C42,具有抑制前列腺癌雄激素非依賴性 PC3M細(xì)胞生長和誘導(dǎo)細(xì)胞凋亡的作用。進(jìn)一步分析發(fā)現(xiàn),其對(duì)PC3M誘導(dǎo)的細(xì)胞凋亡為caspase依賴性的凋亡,C42處理細(xì)胞后,細(xì)胞的caspase-3/7的活性增加,PARP的被剪切程度增強(qiáng)。這對(duì)利用C42類化合物作為腫瘤藥物的臨床應(yīng)用所涉及的基礎(chǔ)研究具有重要意義。
我們研究發(fā)現(xiàn),在利用0.25 μmol/L C42處理PC3M細(xì)胞6 h后,即可顯著增加caspase-3/7的活性,增加 PARP的被剪切量,顯著誘導(dǎo)caspase依賴的細(xì)胞凋亡發(fā)生;處理12 h后,即可顯著降低細(xì)胞增殖能力,抑制PC3M細(xì)胞的生長;24 h后,與對(duì)照相比,其細(xì)胞增殖抑制率可達(dá)至36%。當(dāng)C42使用濃度提高至0.5 μmol/L時(shí),處理6 h后,其不僅能顯著地增加早期凋亡細(xì)胞的數(shù)量,且可顯著提高晚期凋亡細(xì)胞和/或壞死細(xì)胞的比例;處理12 h后,其細(xì)胞增殖抑制率可達(dá)44%。
而目前篩選出的對(duì) PC3M細(xì)胞具有殺傷作用的其他化合物,如金絲桃素在 1 μmol/L濃度下,處理48 h后,其細(xì)胞增殖抑制率為20%[7];羥基喜樹堿在2.74 μmol/L濃度下,處理24 h后,其抑制率為26.57%[8];苦參堿在503 μmol/L濃度下,處理24 h后,其抑制率為1.68%[9];DIM在10 μmol/L濃度下,處理48 h后,其抑制率為35.6%[6]。由此可見,C42對(duì)PC3M細(xì)胞具有更好的殺傷作用,且具有更高的潛在應(yīng)用價(jià)值。
目前研究認(rèn)為,促使細(xì)胞導(dǎo)向死亡的途徑主要有3種,即細(xì)胞凋亡 (Apoptosis)、自噬性死亡(Autophagic cell death) 和細(xì)胞壞死 (Necrosis)[19]。C42處理對(duì)PC3M細(xì)胞生長的抑制,除通過細(xì)胞凋亡途徑使細(xì)胞導(dǎo)向死亡外,是否還通過引起自噬性細(xì)胞死亡、細(xì)胞壞死而使細(xì)胞導(dǎo)向死亡,C42是否可抑制實(shí)體瘤的生長等,這些對(duì)進(jìn)一步開發(fā)利用ETPs類真菌來源的活性化合物和研究此類化合物的可能臨床治療前列腺癌均十分重要,有待進(jìn)一步深入研究。
致謝 衷心感謝中國科學(xué)院微生物研究所姜學(xué)軍研究員給予本文的指導(dǎo)與幫助!
[1] Hua LX, Qiao D, Xu B, et al. Clinical and pathological characteristics of screen-detected versus clinically diagnosed prostate cancer in Nanjing, China. Med Oncol, 2011, 28(1): 357?364. [2] Fang RK. The study of environment and cancer in China-with special reference to Shanghai. [2004-01-25]. http://www.geographie.uni-muenchen.de/ geomed/pdf_presentations/07_Sat_Rukang.pdf.
[3] Soriano-Hernández AD, Galvan-Salazar HR, Montes-Galindo DA, et al. Antitumor effect of meclofenamic acid on human androgenindependent prostate cancer: a preclinical evaluation. Int Urol Nephrol, 2011: 1?7.
[4] Miyamoto H, Messing E M, Chang C. Androgen deprivation therapy for prostate cancer: current status and future prospects. Prostate, 2004, 61(4): 332?353.
[5] Isaacs JT. New strategies for the medical treatment of prostate cancer. BJU Int, 2005, 96(S2): 35?40.
[6] Pan YZ, Li Y, Zhao YY, et al. Inhibitory effect of DIM on growth of human prostate cancer cell PC3M. J Jilin Univ: Med Ed, 2006, 32(6): 1034?1036.潘玉琢, 李揚(yáng), 趙燕潁, 等. DIM對(duì)人激素非依賴性前列腺癌細(xì)胞PC3M的生長抑制作用. 吉林大學(xué)學(xué)報(bào): 醫(yī)學(xué)版, 2006, 32(6): 1034?1036.
[7] Gao LF, Meng Y, Zhao XJ, et al. Inhibitory effect of photoactivated hypericin on growth of human prostate cancer cell PC3M. J Jilin Univ: Med Ed, 2004, 30(1): 97?99.高麗芳, 孟艷, 趙雪儉, 等. 光活化的金絲桃素對(duì)人前列腺癌細(xì)胞PC3M的生長抑制作用. 吉林大學(xué)學(xué)報(bào): 醫(yī)學(xué)版, 2004, 30(1): 97?99.
[8] Jin GH, Chen S, Yang L, et al. Hydroxycamptothecin induced human prostate cancer Line PC-3M cell apoptosis. Chin J Lab Diagn, 2010, 14(1): 12?13.金光虎, 陳爽, 楊麗, 等. 羥基喜樹堿對(duì)人前列腺癌 PC-3M細(xì)胞凋亡的影響. 中國實(shí)驗(yàn)診斷學(xué), 2010, 14(1): 12?13.
[9] Jin GH, Gao J, Mao XQ, et al. Matrine induced human prostate cancer Line PC-3M cell apoptosis and affected the Osteopontin levels. Chin J Gerontol, 2009, 29(21): 2758?2759.金光虎, 高吉, 毛小強(qiáng), 等. 苦參堿誘導(dǎo)人前列腺癌 pc-3M 細(xì)胞凋亡及對(duì)骨橋蛋白表達(dá)的影響.中國老年學(xué)雜志, 2009, 29(21): 2758?2759.
[10] Mao XQ, Na W L, Zhao D, et al. Effects of tea polyphenol on proliferation inhibition and apoptosis in human prostate cancer PC-3M cells. Chin J Lab Diagn, 2010, 14(2): 170?173.毛小強(qiáng), 那萬里, 趙丹, 等. 茶多酚對(duì)前列腺癌PC-3M 細(xì)胞增殖與凋亡的影響. 中國實(shí)驗(yàn)診斷學(xué), 2010, 14(2): 170?173.
[11] Dong JY, He HP, Shen YM, et al. Nematicidal epipolysulfanyldioxopiperazines from Gliocladium roseum. J Nat Prod, 2005, 68(10): 1510?1513.
[12] Zhang N, Chen YL, Jiang RX, et al. PARP and RIP 1 are required for autophagy induced by 11'-deoxyverticillin A, which precedes caspase-dependent apoptosis. Autophagy, 2011, 7(6): 598?612.
[13] Schlegel J, Peters I, Orrenius S, et al. CPP32/Apopain is a key interleukin 1β converting enzyme-like protease involved in Fas-mediated apoptosis. J Biol Chem, 1996, 271(4): 1841?1844. [14] Liu XS, Kim CN, Yang J, et al. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell, 1996, 86(1): 147?157.
[15] Gardiner DM, Waring P, Howlett BJ. The epipolythiodioxopiperazine (ETP) class of fungal toxins: distribution, mode of action, functions and biosynthesis. Microbiology, 2005, 151(4): 1021?1032.
[16] Bernardo PH, Brasch N, Chai CLL, et al. A novel redox mechanism for the glutathione-dependent reversible uptake of a fungal toxin in cells. J Biol Chem, 2003, 278(47): 46549?46555.
[17] Beaver JP, Waring P. Lack of correlation between early intracellular calcium ion rises and the onset of apoptosis in thymocytes. Immunol Cell Biol, 1994, 72(6): 489?499.
[18] Yanagihara M, Sasaki-Takahashi N, Sugahara T, et al. Leptosins isolated from marine fungus Leptoshaeria species inhibit DNA topoisomerases I and/or II and induce apoptosis by inactivation of Akt/protein kinase B. Cancer Sci, 2005, 96(11): 816?824.
[19] Kroemer G, Galluzzi L, Vandenabeele P, et al. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ, 2009, 16(1): 3?11.