6′-羟基爵床定A对肿瘤细胞的抑制活性及其对肿瘤细胞氧化还原系统的影响

目的筛选对6′-羟基爵床定A(JR6)敏感的肿瘤细胞株并探讨其对细胞氧化还原功能的影响。方法将人膀胱癌细胞株EJ、肝癌细胞Bel、肺癌细胞A549、结肠癌细胞HCT-8和HT-29、胃癌细胞BGC、大肠癌细胞LS180、宫颈癌细胞HeLa、肝癌细胞HepG2以及乳腺癌细胞MCF-7分为正常对照组、阳性药对照顺铂、多柔比星、替尼泊苷、依托泊苷和5-氟尿嘧啶组及JR6组,细胞加入相应的药物培养48h后,采用噻唑蓝比色法(MTT)检测细胞存活率并计算IC50值。选用对JR6与多柔比星作用敏感的人膀胱癌细胞EJ,加入相应的药物培养48h后,分别检测细胞内超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和过氧化氢酶(CAT)活性及丙二醛(MDA)含量。EJ细胞分为外源性SOD+多柔比星9.19μmol.L-1组和外源性SOD+JR63.02,9.07,27.2,81.7和245μmol.L-1处理组,其中SOD分别为0,1.9,5.6,16.7,50和150kU.L-1,MTT法检测细胞存活率。多柔比星9.19μmo.lL-1,JR612.3,49.0和196μmo.lL-1加入EJ细胞,24h后用激光共聚焦显微镜检测EJ细胞内活性氧类的含量。结果 EJ细胞对JR6较为敏感,IC50为57.1μmol.L-1,多柔比星对EJ细胞的IC50为4.08μmol.L-1。其余9种肿瘤细胞对JR6的IC50值为71.3～123μmol.L-1。与正常对照组比较,多柔比星9.19μmo.lL-1能明显降低EJ细胞内SOD,GSH-Px和CAT活性,分别降低了(64.3±2.1)%,(66.5±3.5)%和(49.6±1.9)%,而MDA的含量明显升高了(432.0±5.4)%(P<0.01);JR63.02,9.07,27.2,81.7和245μmo.lL-1使EJ细胞内SOD的活性分别降低了(7.28±0.3)%,(57.1±3.2)%,(66.5±4.7)%,(72.1±5.5)%和(77.8±2.4)%;GSH-Px活性分别降低了(20.3±1.6)%,(32.8±2.3)%,(45.3±3.6)%,(59.3±4.5)%和(71.9±4.2)%,CAT活性分别降低了(10.1±0.6)%,(15.9±0.7)%,(25.9±2.3)%,(38.8±3.5)%和(52.4±3.9)%;同时MDA含量分别升高了(24.4±1.3)%,(90.2±8.70)%,(217.0±19.0)%,(356.0±24.0)%和(539.0±32.0)%(P<0.05,P<0.01)。与无外源性SOD组比较,在外源性SOD存在时,多柔比星与JR6对EJ细胞的生长抑制率明显下降(P<0.05)。与正常对照组相比,多柔比星9.19μmol.L-1使EJ细胞活性氧类水平升高了(43.0±2.1)%,JR612.3,49.0和196μmol.L-1使EJ细胞ROS水平分别升高了(40.7±0.7)%,(84.1±6.3)%和(151.0±2.9)%(P<0.01)。结论人膀胱癌细胞株EJ对JR6最敏感;JR6通过干预细胞内氧化还原系统平衡抑制细胞的增殖,外源性SOD可以拮抗JR6对敏感细胞增殖的抑制作用。

Effect of 6'-hydroxy justicidin A on cell proliferation and redox system in tumor cells

OBJECTIVE To screen 6′-hydroxy justicidin A（JR6）-sensitive tumor cells and explore the cell redox function. METHODS The human bladder cancer cells EJ, human liver cancer cells Bel, human lung cancer cells A549, human colon cancer cells HCT-8 and HT-29, human gastric cancer cells BGC, human colorectal cancer cells LS180, human cervical cancer cells HeLa, human hepatoma cells HepG2, and human breast cancer cells MCF-7 were divided into normal control group, positive control cisplatin, doxorubicin, teniposide, etoposide, 5-fluorouracil groups and JR6 group. The effect of JR6 and positive drugs on inhibition in 10 tumor cell lines for 48 h was measured using MTT method and the values of IC₅₀ were calculated by the methods of curvilinear regression. After EJ cells were cultured with JR6 3.02, 9.07, 27.2, 81.7 and 245 μmol·L^-1 for 48 h, the activities of superoxide dismutase (SOD), glutathion peroxidase (GSH-Px), and catalase (CAT) as wall as the content of malondialdehyde (MDA) in EJ cells were detected. EJ cells were divided into exogenous SOD+doxorubicin 9.19 μmol·L^-1 and exogenous SOD+JR6 3.02, 9.07, 27.2, 81.7 and 245 μmol·L^-1 groups. Proliferation of sensitive cells was measured by MTT method. After EJ cells were cultured with doxorubicin 9.19 μmol·L^-1 and JR6 12.3, 49.0 and 196 μmol·L^-1 for 24 h, the generation of reactive oxygen species in JR6-sensitive tumor cells was measured by laser scanning confocal microscopy. RESULTS MTT assay results showed EJ cells were sensitive to JR6, and IC₅₀ of JR6 in EJ cells was 57.1 μmol·L^-1, while IC₅₀ of JR6 in other tumor cells ranged from 71.3 to 124 μmol·L^-1. Compared with normal control group, doxorubicin 9.19 μmol·L^-1 decreased activities of SOD by (64.3±2.1)%, GSH-Px by (66.5±3.5)% and CAT by (49.6±1.9)%, but increased the content of MDA by (432±5.4)% in EJ cells(P<0.01). JR6 3.02, 9.07, 27.2, 81.7 and 245 μmol·L^-1 significantly decreased activities of SOD (7.28±0.3)%, (57.1±3.2)%, (66.5±4.7)%, (72.1±5.5)% and (78.7±2.4)%, decreased GSH-Px (20.3±1.6)%, (32.8±2.3)%, (45.3±3.6)%, (59.3±4.5)% and (71.9±4.2)%, decreased CAT (10.1±0.6)%, (15.9±0.7)%, (25.9±2.3)%, (38.8±3.5)%, (52.4±3.9)% in EJ cells, respectively; and JR6 3.02, 9.07, 27.2, 81.7 and 245 μmol·L^-1 raised the content of MDA (24.4±1.3)%, (90.2±8.7)%, (217.0±19.0)%, (356.0±24.0)% and (539.0±32.0)%, respctively. Exogenous SOD could protect against the inhibition effect of doxorubicin 9.19 μmol·L^-1 and JR6 3.02, 9.07, 27.2, 81.7 and 245 μmol·L^-1 on EJ cells, compared with no exogenous SOD group (P<0.05). Compared with normal control group, doxorubicin 9.19 μmol·L^-1 increased content of reactive oxygen species by (43.0±2.1)%, JR6 12.3, 49.0 and 196 μmol·L^-1 increased contents of reactive oxygen species by (40.7±0.7)%, (84.1±6.3)% and (151.0±2.9)%, respectively. CONCLUSION The human bladder cancer cell line EJ is JR6-sensitive. JR6 interferes the intracellular balance of redox system to inhibit proliferation of EJ cells. Exogenous SOD can protect against the inhibition effect of JR6 in EJ cells.