DATS逆转K562/A02细胞耐药的机制研究
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摘要
研究目的
急性白血病是一种严重危害人类健康的血液肿瘤。恶性肿瘤表现出来的耐药性一直是治疗失败的主要原因。其中P-gp介导的MDR是肿瘤耐药性的主要机制。逆转MDR可以使化疗失败的机率下降,具有很高的临床价值,已成为目前国内外化疗药物研究的重要方向之一。已发现的多种逆转剂缺乏肿瘤细胞识别特异性,会导致化疗药物药动学发生改变,增加毒性,如药物清除率下降、半衰期延长、血液浓度增高、分配体积增加等,这些都会增加对正常组织的毒性。植物来源的药物资源丰富,作用靶点多,具有高效低毒的优点,显示其在逆转MDR方面有较好的应用前景。本研究应用大蒜素提取物DATS处理K562/A02细胞,观察其对阿霉素的耐药逆转作用,评价DATS与小剂量维拉帕米合用逆转耐药的效果极其毒性作用,并就其逆转作用的分子机制进行深入探讨。
研究内容和方法
1.甲基四唑蓝(MTT)法增殖抑制实验
1.1测定DATS(?)VRP对细胞的毒性作用,药物对细胞生长抑制率(%)=(1-实验孔A值/对照孔A值)×100%,根据线性回归方程求得10%的细胞生长受到抑制所需药物浓度(IC10)。选取DATS和VRP分别低于各自IC10的一个浓度,作为下述实验所需安全浓度,并测定二者以此浓度联合应用对K562/A02细胞生长的抑制率。
1.2测定不同剂量的DATS联合阿霉素(ADM)对K562/A02细胞抑制率的量效和时效关系对照组加入终浓度为5ug/ml的ADM,实验组加入终浓度为5ug/ml的ADM和不同浓度的DATS,培养24、48、72小时后,上酶标仪测定A值。计算不同作用时间不同剂量的DATS和ADM联合作用对细胞的生长抑制率。
1.3测定逆转剂对细胞药物敏感性的影响：加入不同浓度ADM培养48小时后,计算阿霉素对对照组和各加药组细胞的半数抑制浓度(IC50)。耐药倍数=耐药株IC50/敏感株IC50;逆转倍数=耐药株IC50/加逆转剂后IC50。
2.光镜下细胞形态的改变取对数生长期的K562/A02细胞制成浓度为1×105/mL的细胞悬液,接种于预先铺置无菌盖玻片的24孔培养板内,分组为：
①K562/A02,
②K562/A02+ADM
③K562/A02+DATs+ADM
②、③组内均加入终浓度ADM1μg/mL,③组加入IC10浓度的DATS.在37℃,5%C02,及饱和湿度下培养24、48、72小时后,用HE法染色,光镜下观察,拍照。
3.流式细胞术检测细胞内ADM浓度
取对数生长期的K562和K562/A02细胞,实验分组：
①K562
②K562/A02
③K562+ADM
④K562/A02+ADM
⑤K562/A02+DATS+ADM
⑥K562/A02+VRP+ADM
⑦K562/A02+DATS+VRP+ADM
以⑥组作为阳性对照,以④组作为阴性对照.DATS和VRP终浓度为IC10所需浓度。调整各组细胞浓度为1×105/mL,分别加入DATS或VRP培养48h后,③-⑦加入终浓度为5μg/mL的ADM,再共同培养2小时利用ADM自身荧光的特性,经FCM检测对照组和各加药组细胞内ADM荧光强度。
4.流式细胞术检测细胞膜P-gp
取对数生长期的K562和K562/A02细胞,使其浓度为1×105/mL,分为
①K562组
②K562/A02组
③K562/A02+DATS组
④K562/A02+VRP组
⑤K562/A02+DATS+VRP组
每组平行3瓶。DATS和VRP终浓度为IC10所需浓度。对照组和各加药组细胞在37℃,5%C02,及饱和湿度下培养48小时。加入藻红蛋白(PE)标记的鼠抗人P-gp单抗P-gp-PE和同型对照IgG2a-PE6μ1,用流式细胞术检测细胞膜P-gp。
5.流式细胞术检测细胞凋亡
取对数生长期的K562/A02细胞,使其浓度为1×105/mL,；接种于50ml培养瓶中。分组：
①K562/A02组
②K562/A02+DATS组
③K562/A02+VRP组
④K562/A02+DATS+VRP组
每组平行3瓶。DATS和VRP终浓度为IC10所需浓度。对照组和各加药组细胞在37℃、5%CO2、饱和湿度下培养48小时。加5μlAnnexin V/FITC和10μ1(20μg/ml)的PI溶液,用流式细胞仪检测细胞凋亡,FACS软件分析数据。右下象限是Annexin V+PI一细胞为早期凋亡细胞。
6. RT-PCR检测基因的改变
取对数生长期K562/A02细胞,分别用含10%灭活新生牛血清的RPMI-1640稀释成1×105/mL,接种于50ml培养瓶中,实验分为
①K562组
②K562/A02组
③K562/A02+DATS组
④K562/A02+VRP组
⑤K562/A02+DATS+小剂量VRP组
⑥K562/A02+DATS+VRP组,
每组平行3瓶。DATS和VRP终浓度为IC10所需浓度。小剂量VRP终浓度为1/2IC10所需浓度。在37℃,5%CO2,及饱和湿度下培养48小时。采用半定量逆转录聚合酶链反应,检测各组细胞多药耐药基因mdrl及凋亡相关基因Bcl-2. Bax、 Caspase-3,和NF-K B/p65、IκBα的mRNA表达
7. Western blot检测蛋白在细胞膜上的表达取对数生长期K562/A02细胞,分别用含10%灭活新生牛血清的RPMI-1640稀释成1×105/mL,接种于50ml培养瓶中,实验分为：
①K562组
②K562/A02组
③K562/A02+DATS组
④K562/A02+VRP组
⑤K562/A02+DATS+小剂量VRP组
⑥K562/A02+DATS+VRP组
每组平行3瓶。DATS和VRP终浓度为IC10所需浓度。小剂量VRP终浓度为1/2IC10所需浓度。在37℃,5%C02,及饱和湿度下培养48小时。各组细胞提取细胞总蛋白,经SDS-PAGE聚丙烯酰胺凝胶电泳,转膜,封闭,以鼠抗人Mdr-1抗体、兔抗人NF-K B/p65抗体、鼠抗人IκBα抗体、兔抗人Caspase-3蛋白单克隆抗体作为一抗,二抗为HRP(辣根过氧化物酶)标记的羊抗兔IgG,,DAB显色,扫描结果,以密度值反应蛋白表达量。
9.统计学处理
实验数据以均数±标准差表示,采用SPSS17.0软件进行统计学分析处理。实验组与对照组之间采用t检验,多组间的比较采用单向方差分析的方法。析因设计资料采用双因素分析方法,并绘制交互作用图,若两条线是平行的,则说明没有交互作用。P<0.05则认为差异有统计学意义。
结果
1.MTT检测结果
DATS和VRP对K562/A02细胞的IC10分别为2.31±0.22μmol/L和4.03±0.13μg/m1。选择DATS2μmol/L、VRP4μg/ml作为安全范围药物剂量用于下面的实验。以此浓度的DATS和VRP联合作用于K562/A02细胞,培养48小时后,细胞的生存率为：92.72±3.26%。两药合用毒性并无相加。
不同剂量的DATS联合阿霉素(ADM)对K562/A02细胞抑制率的具有量效和时效关系。
DATS单药时对K562/A02细胞耐药逆倍数为3.789,VRP单药时对K562/A02细胞耐药逆倍数为12.31,联合应用(2μg/m1)VRP和DATS的逆转倍数为12.21,联合应用(4μg/m1)VRP和DATS的逆转倍数为34.38,证明DATS和VRP联合应用可以增加逆转作用,且小剂量VRP和DATS联合应用可以达到大剂量VRP单药的逆转效果。
2.光镜下细胞形态的改变
未加入逆转剂时,K562/A02细胞增殖旺盛,核分裂像多见。单独应用ADM(1μg/mL)作用48小时,K562/A02细胞增殖率抑制不明显。K562/A02+ADM+DATS组培养24小时,细胞数目明显减少,核分裂像少见。继续培养48小时后,部分细胞被裂解成数个大小不等的小体,细胞体积缩小,染色质凝聚附在核膜周边,嗜碱性增强,细胞核固缩呈均一的致密物,可见凋亡小体,部分核碎裂。72小时后,细胞数目更少,凋亡细胞比例增加。
3.流式细胞术检测细胞内ADM浓度：K562细胞和K562/A02细胞内自身荧光强度很弱,分别为0.36±0.13和0.59±0.04。K562细胞经ADM处理后,细胞内ADM荧光强度为4.24±0.15,K562/A02细胞经ADM处理后,细胞内ADM荧光强度为2.49±0.27,与K562细胞比较有显著性差异(P<0.01)。加入DATS或VRP后,K562/A02细胞中的ADM荧光强度比加药前明显增强(P<0.01)。各加药组间相比无显著性差异。
4.流式细胞术检测细胞膜P-gp：K562细胞P-gp表达率极低(0.53±0.07),K562/A02细胞P-gp表达率高(9.16±1.27),与K562细胞有显著性差异(P<0.01)。加入逆转剂DATS或VRP后,K562/A02细胞的P-gp表达率明显降低(P<0.01)。各加药组间无显著性差异。
5.流式细胞术检测细胞凋亡K562/A02细胞的自然凋亡率是0.90±0.17%,加入DATS后凋亡率为12.15±0.78%,加入VRP后凋亡率为11.55±1.91%,两药合用时凋亡率为12.55±0.64K562/A02组和各加药组间均有显著性差异(P=0.000)。各加药组间无显著性差异。
6. RT-PCR检测基因的改变
K562/A02组的NF-KB/p65表达明显强于K562组(P<0.05)。各加药组均可以明显减弱K562/A02细胞的NF-KB/p65表达。(P<0.01)。K562组细胞的IκBα表达明显高于K562/A02组细胞。DATS、VRP单独使用对提高K562/A02组细胞IκBα的表达作用不明显(P=0.4,0.133)。两药合用效果优于单药(P<0.05)。DATS对Bcl-2(P=0.166)、Bax(P=0.062)的表达作用不明显。K562/A02细胞mdr-1表达明显高于K562细胞(P=0.000)。各加药组均可明显减弱K562/A02细胞的mdr-1表达(P<0.05)。K562细胞Caspase_3表达明显高于K562/A02细胞(P=0.000),各加药组均可以明显增加K562/A02细胞的Caspase_3表达(P<0.01)。
7. Western blot检测K562/A02细胞膜上蛋白表达的变化：
K562细胞NF-KB/p65表达率明显弱于K562/A02细胞(P<0.01),各加药组均可以明显减弱K562/A02细胞的NF-κB/p65表达(P<0.01)。K562细胞IκBα表达明显强于K562/A02细胞(P<0.05)。DATS、VRP单用对IκBα增强作用不明显(P=0.445,0.117),两药合用效果优于单药(P<0.05)。K562细胞mdr-1表达明显弱于K562/A02细胞(P=0.000)。各加药组均可明显减弱K562/A02细胞的mdr-1表达(P<0.05)。K562细胞的Caspase_3表达明显高于K562/A02细胞(P<0.05),各加药组均可以明显增加K562/A02细胞的Caspase_3表达(P<0.05)。
结论
1.大蒜素DATS具有逆转K562/A02细胞耐药的作用,与VRP合用时具有协同作用。
2. DATS的这种逆转作用是通过减弱mdr-1基因的表达减少化疗药物外排和激活Caspase-3表达促进凋亡而实现的。
3. NF-κB通路参与了上述基因的调节过程。
Objectives
The multidrug resistance (MDR) of leukemic cells to chemotherapy remains the most significant cause of treatment failure in acute leukemia. A number of studies have shown that the major contributor to MDR is increased drug efflux mediated by P-glycoprotein (P-gp), a product of the mdr-1gene. Many plant-derived drugs or herbal formulations have been proved have anti-tumor potential in vitro and in vivo. Diallyl trisulfide (DATS) is the main sulfuric compound in garlic. Our goal was to explore whether diallyl trisulfide (DATS) could overcome P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in MDR K562/A02cells, and to prove whether NF-κB activation is involved in the MDR reversal mechanism of DATS.
Content and Methods
1. Assay of in vitro drug sensitivity
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay was used to compare the MDR of the K562and K562/A02cells to adriamycin. The absorbance value was measured with a spectrophotometer at the wavelength of570nm. IC50of the drugs was calculated based on the MTT assay. Inhibition of cell viability=(1-average A value of experimental group/average A value of blank control group)×100%. Drug resistance fold=IC50of drug-resistant group/IC50of sensitive group. Reverse fold=IC50before reversal/IC50after reversal
2. Observation of morphological changes by light microscopy
The morphological changes of apoptotic cells with Hematoxylin-Eosin (HE) staining were observed by light microscopy. K562/A02cells were grown at1×105cells/ml in a complete RPMI1640medium on a24-well plate with a coverslip set at the bottom, treatment with2μmol/L DATS combined with a final concentration of1 mg/L adriamycin for24h to72h,(treatment with adriamycin alone as control). Finally, the specimen was observed by light microscopy.
3. Detection of intracellular adriamycin concentration by FCM
The adriamycin concentration in K562/A02cells was measured by flow cytometry (FCM). K562/A02cells were treated with2μmol/L DATS or4mg/L verapamil and incubated for48h. A total of10000gated cells were detected for each sample, which were analyzed by Modfit LT software.
4. Detection of P-gp expression by FCM
P-gp expression on the surface membrane of K562/A02cells was determined by a direct immunoflourescence staining technique. K562/A02cells were co-cultured with2μmol/L DATS or4mg/L verapamil for48h. Protein expression was analyzed using Cell Quest software.
5.Apoptosis assay by statistical FCM
The apoptosis rates were measured using flow cytometric assay. Cell labeling was performed using annexin V conjugated to FITC, which binds to phosphatidylserine exposed on the surface membrane of cells undergoing apoptosis. The samples were measured using a flow cytometer with FACS software.
6.Semi-quantitative RT-PCR assay
After treatment with the drugs (2μmol/L DATS or4mg/L verapamil), in vitro total mRNA was extracted from the cells with Trizol reagent according to the manufacturer's instructions. The products were identified by electrophoresis using1.5%agarose gel. Using β-actin as internal reference, the products were further analyzed using Alpha gel image analysis system.
7.Western blot analysis of protein expression
After treatment with the drugs (2μmol/L DATS or4mg/L verapamil), total protein was isolated and subjected to sodium dodecyl sulfate PAGE analysis and transferred to a polyvinylidene difluoride membrane. After normalization by the corresponding β-actin expression, protein expression level was determined by densitometry scans and measured with Quantity One software.
8.Analyses
Statistical calculations were carried out with SPSS17.0for Windows software package. The results were expressed as mean±standard deviation of three independent experiments. Student's t-test was used for the statistical analyses, and P values<0.05were considered significant. The synergetic effect of the two drugs was analyzed using factorial analysis.
Results
1. Drug sensitivity
MTT assay was used to study the cytotoxicity of adriamycin. The ability of DATS at2μmol/L to enhance the cytotoxicity of adriamycin in K562/A02was examined. The IC50value of adriamycin for K562/A02decreased after treatment with DATS. The time-and concentration-dependent reversal effects of DATS on the K562/A02cells were observed for24,48, and72h. The higher the concentration of DATS used, the better the inhibitive effect.
2. Apoptosis observed by light microscopy
After simultaneous treatment of adriamycin (1μg/mL) with DATS (2μmol/L) for24h, the proliferation of K562/A02cells slowed down. After48h, K562/A02cells showed cell shrinkage, chromatin condensation, margination, nuclear fragmentation, apoptotic bodies, and typical apoptotic cytomorphological features. After72h, more apoptotic cells and less surviving K562/A02cells were detected.
3. Detection of intracellular adriamycin concentration
The effect of DATS on the intracellular accumulation of adriamycin was examined by FCM. After treatment with DATS, adriamycin fluorescence intensity in K562/A02cells increased to4.38±1.08, which there was a significant difference compared to that without DATS-treatment cells(P<0.01). These results showed that DATS had ability to enhance the intracellular concentration of adriamycin.
4. Alteration of P-gp expression
DATS-treated K562/A02cells were incubated with phycoerythrin-conjugated UIC2, and then detected by FCM. The expression of P-gp in K562was lower than that in K562/A02(P<0.01). After treatment with DATS or verapamil in K562/A02cells, P-gp expression decreased.
5. Apoptosis statistical FCM assay
Apoptosis of K562/A02cells was induced by DATS or verapamil. After incubation with either DATS (2μmol/L) or verapamil (4μg/ml) for48h, apoptotic percentages of K562/A02cells were12.15±0.78%and11.55±1.91%, respectively. Evident differences were found compared with the control (0.9±0.17%,P=0.000).
6. Detection of gene expression
As demonstrated by semi-quantitative RT-PCR, overexpression of mdrl mRNA was detected in K562/A02cells compared with K562cells (P=0.000). DATS could downregulate the expression of mdrl and NF-κB/p65(P<0.05) and upregulate the expression of caspase-3(P<0.05). However, DATS cannot evidently increase the expression level of IκBα, Bcl-2and Bax.(P>0.05)
7. Western blot analysis of protein expression
The Western blot report revealed that the expression of mdrl protein ai NF-KB/p65protein were much higher in K562/A02cells compared with K562ce (P=0.000). And the expression level of IκBα protein and caspase-3protein K562/A02cells was much lower than that of K562cells(P<0.05). DATS cou downregulate the expression of mdrl and NF-KB/p65(P<0.05) and upregulate t expression of caspase-3(P<0.05). However, DATS cannot evidently increase t expression level of IκBα(P>0.05)
Conclusion
The present study has demonstrated that DATS can serve as a novel, non-toxic modulator of MDR and can reverse the MDR of K562/A02cells in vitro by increasing intracellular adriamycin concentration, downregulating mdr-1expression, and inducing apoptosis by activating increased caspase-3expression. We therefore conclude that DATS can block NF-κB activation, which produces the downstream inhibitory effects on chemotherapy sensitivity and apoptosis of K562/A02cells. DATS could be a highly feasible candidate for the development of a new MDR reversal agent.
急性白血病是一种严重危害人类健康的血液肿瘤。恶性肿瘤表现出来的耐药性一直是治疗失败的主要原因。其中P-gp介导的MDR是肿瘤耐药性的主要机制。逆转MDR可以使化疗失败的机率下降,具有很高的临床价值,已成为目前国内外化疗药物研究的重要方向之一。已发现的多种逆转剂缺乏肿瘤细胞识别特异性,会导致化疗药物药动学发生改变,增加毒性,如药物清除率下降、半衰期延长、血液浓度增高、分配体积增加等,这些都会增加对正常组织的毒性。植物来源的药物资源丰富,作用靶点多,具有高效低毒的优点,显示其在逆转MDR方面有较好的应用前景。本研究应用大蒜素提取物DATS处理K562/A02细胞,观察其对阿霉素的耐药逆转作用,评价DATS与小剂量维拉帕米合用逆转耐药的效果极其毒性作用,并就其逆转作用的分子机制进行深入探讨。
研究内容和方法
1.甲基四唑蓝(MTT)法增殖抑制实验
1.1测定DATS(?)VRP对细胞的毒性作用,药物对细胞生长抑制率(%)=(1-实验孔A值/对照孔A值)×100%,根据线性回归方程求得10%的细胞生长受到抑制所需药物浓度(IC10)。选取DATS和VRP分别低于各自IC10的一个浓度,作为下述实验所需安全浓度,并测定二者以此浓度联合应用对K562/A02细胞生长的抑制率。
1.2测定不同剂量的DATS联合阿霉素(ADM)对K562/A02细胞抑制率的量效和时效关系对照组加入终浓度为5ug/ml的ADM,实验组加入终浓度为5ug/ml的ADM和不同浓度的DATS,培养24、48、72小时后,上酶标仪测定A值。计算不同作用时间不同剂量的DATS和ADM联合作用对细胞的生长抑制率。
1.3测定逆转剂对细胞药物敏感性的影响：加入不同浓度ADM培养48小时后,计算阿霉素对对照组和各加药组细胞的半数抑制浓度(IC50)。耐药倍数=耐药株IC50/敏感株IC50;逆转倍数=耐药株IC50/加逆转剂后IC50。
2.光镜下细胞形态的改变取对数生长期的K562/A02细胞制成浓度为1×105/mL的细胞悬液,接种于预先铺置无菌盖玻片的24孔培养板内,分组为：
①K562/A02,
②K562/A02+ADM
③K562/A02+DATs+ADM
②、③组内均加入终浓度ADM1μg/mL,③组加入IC10浓度的DATS.在37℃,5%C02,及饱和湿度下培养24、48、72小时后,用HE法染色,光镜下观察,拍照。
3.流式细胞术检测细胞内ADM浓度
取对数生长期的K562和K562/A02细胞,实验分组：
①K562
②K562/A02
③K562+ADM
④K562/A02+ADM
⑤K562/A02+DATS+ADM
⑥K562/A02+VRP+ADM
⑦K562/A02+DATS+VRP+ADM
以⑥组作为阳性对照,以④组作为阴性对照.DATS和VRP终浓度为IC10所需浓度。调整各组细胞浓度为1×105/mL,分别加入DATS或VRP培养48h后,③-⑦加入终浓度为5μg/mL的ADM,再共同培养2小时利用ADM自身荧光的特性,经FCM检测对照组和各加药组细胞内ADM荧光强度。
4.流式细胞术检测细胞膜P-gp
取对数生长期的K562和K562/A02细胞,使其浓度为1×105/mL,分为
①K562组
②K562/A02组
③K562/A02+DATS组
④K562/A02+VRP组
⑤K562/A02+DATS+VRP组
每组平行3瓶。DATS和VRP终浓度为IC10所需浓度。对照组和各加药组细胞在37℃,5%C02,及饱和湿度下培养48小时。加入藻红蛋白(PE)标记的鼠抗人P-gp单抗P-gp-PE和同型对照IgG2a-PE6μ1,用流式细胞术检测细胞膜P-gp。
5.流式细胞术检测细胞凋亡
取对数生长期的K562/A02细胞,使其浓度为1×105/mL,；接种于50ml培养瓶中。分组：
①K562/A02组
②K562/A02+DATS组
③K562/A02+VRP组
④K562/A02+DATS+VRP组
每组平行3瓶。DATS和VRP终浓度为IC10所需浓度。对照组和各加药组细胞在37℃、5%CO2、饱和湿度下培养48小时。加5μlAnnexin V/FITC和10μ1(20μg/ml)的PI溶液,用流式细胞仪检测细胞凋亡,FACS软件分析数据。右下象限是Annexin V+PI一细胞为早期凋亡细胞。
6. RT-PCR检测基因的改变
取对数生长期K562/A02细胞,分别用含10%灭活新生牛血清的RPMI-1640稀释成1×105/mL,接种于50ml培养瓶中,实验分为
①K562组
②K562/A02组
③K562/A02+DATS组
④K562/A02+VRP组
⑤K562/A02+DATS+小剂量VRP组
⑥K562/A02+DATS+VRP组,
每组平行3瓶。DATS和VRP终浓度为IC10所需浓度。小剂量VRP终浓度为1/2IC10所需浓度。在37℃,5%CO2,及饱和湿度下培养48小时。采用半定量逆转录聚合酶链反应,检测各组细胞多药耐药基因mdrl及凋亡相关基因Bcl-2. Bax、 Caspase-3,和NF-K B/p65、IκBα的mRNA表达
7. Western blot检测蛋白在细胞膜上的表达取对数生长期K562/A02细胞,分别用含10%灭活新生牛血清的RPMI-1640稀释成1×105/mL,接种于50ml培养瓶中,实验分为：
①K562组
②K562/A02组
③K562/A02+DATS组
④K562/A02+VRP组
⑤K562/A02+DATS+小剂量VRP组
⑥K562/A02+DATS+VRP组
每组平行3瓶。DATS和VRP终浓度为IC10所需浓度。小剂量VRP终浓度为1/2IC10所需浓度。在37℃,5%C02,及饱和湿度下培养48小时。各组细胞提取细胞总蛋白,经SDS-PAGE聚丙烯酰胺凝胶电泳,转膜,封闭,以鼠抗人Mdr-1抗体、兔抗人NF-K B/p65抗体、鼠抗人IκBα抗体、兔抗人Caspase-3蛋白单克隆抗体作为一抗,二抗为HRP(辣根过氧化物酶)标记的羊抗兔IgG,,DAB显色,扫描结果,以密度值反应蛋白表达量。
9.统计学处理
实验数据以均数±标准差表示,采用SPSS17.0软件进行统计学分析处理。实验组与对照组之间采用t检验,多组间的比较采用单向方差分析的方法。析因设计资料采用双因素分析方法,并绘制交互作用图,若两条线是平行的,则说明没有交互作用。P<0.05则认为差异有统计学意义。
结果
1.MTT检测结果
DATS和VRP对K562/A02细胞的IC10分别为2.31±0.22μmol/L和4.03±0.13μg/m1。选择DATS2μmol/L、VRP4μg/ml作为安全范围药物剂量用于下面的实验。以此浓度的DATS和VRP联合作用于K562/A02细胞,培养48小时后,细胞的生存率为：92.72±3.26%。两药合用毒性并无相加。
不同剂量的DATS联合阿霉素(ADM)对K562/A02细胞抑制率的具有量效和时效关系。
DATS单药时对K562/A02细胞耐药逆倍数为3.789,VRP单药时对K562/A02细胞耐药逆倍数为12.31,联合应用(2μg/m1)VRP和DATS的逆转倍数为12.21,联合应用(4μg/m1)VRP和DATS的逆转倍数为34.38,证明DATS和VRP联合应用可以增加逆转作用,且小剂量VRP和DATS联合应用可以达到大剂量VRP单药的逆转效果。
2.光镜下细胞形态的改变
未加入逆转剂时,K562/A02细胞增殖旺盛,核分裂像多见。单独应用ADM(1μg/mL)作用48小时,K562/A02细胞增殖率抑制不明显。K562/A02+ADM+DATS组培养24小时,细胞数目明显减少,核分裂像少见。继续培养48小时后,部分细胞被裂解成数个大小不等的小体,细胞体积缩小,染色质凝聚附在核膜周边,嗜碱性增强,细胞核固缩呈均一的致密物,可见凋亡小体,部分核碎裂。72小时后,细胞数目更少,凋亡细胞比例增加。
3.流式细胞术检测细胞内ADM浓度：K562细胞和K562/A02细胞内自身荧光强度很弱,分别为0.36±0.13和0.59±0.04。K562细胞经ADM处理后,细胞内ADM荧光强度为4.24±0.15,K562/A02细胞经ADM处理后,细胞内ADM荧光强度为2.49±0.27,与K562细胞比较有显著性差异(P<0.01)。加入DATS或VRP后,K562/A02细胞中的ADM荧光强度比加药前明显增强(P<0.01)。各加药组间相比无显著性差异。
4.流式细胞术检测细胞膜P-gp：K562细胞P-gp表达率极低(0.53±0.07),K562/A02细胞P-gp表达率高(9.16±1.27),与K562细胞有显著性差异(P<0.01)。加入逆转剂DATS或VRP后,K562/A02细胞的P-gp表达率明显降低(P<0.01)。各加药组间无显著性差异。
5.流式细胞术检测细胞凋亡K562/A02细胞的自然凋亡率是0.90±0.17%,加入DATS后凋亡率为12.15±0.78%,加入VRP后凋亡率为11.55±1.91%,两药合用时凋亡率为12.55±0.64K562/A02组和各加药组间均有显著性差异(P=0.000)。各加药组间无显著性差异。
6. RT-PCR检测基因的改变
K562/A02组的NF-KB/p65表达明显强于K562组(P<0.05)。各加药组均可以明显减弱K562/A02细胞的NF-KB/p65表达。(P<0.01)。K562组细胞的IκBα表达明显高于K562/A02组细胞。DATS、VRP单独使用对提高K562/A02组细胞IκBα的表达作用不明显(P=0.4,0.133)。两药合用效果优于单药(P<0.05)。DATS对Bcl-2(P=0.166)、Bax(P=0.062)的表达作用不明显。K562/A02细胞mdr-1表达明显高于K562细胞(P=0.000)。各加药组均可明显减弱K562/A02细胞的mdr-1表达(P<0.05)。K562细胞Caspase_3表达明显高于K562/A02细胞(P=0.000),各加药组均可以明显增加K562/A02细胞的Caspase_3表达(P<0.01)。
7. Western blot检测K562/A02细胞膜上蛋白表达的变化：
K562细胞NF-KB/p65表达率明显弱于K562/A02细胞(P<0.01),各加药组均可以明显减弱K562/A02细胞的NF-κB/p65表达(P<0.01)。K562细胞IκBα表达明显强于K562/A02细胞(P<0.05)。DATS、VRP单用对IκBα增强作用不明显(P=0.445,0.117),两药合用效果优于单药(P<0.05)。K562细胞mdr-1表达明显弱于K562/A02细胞(P=0.000)。各加药组均可明显减弱K562/A02细胞的mdr-1表达(P<0.05)。K562细胞的Caspase_3表达明显高于K562/A02细胞(P<0.05),各加药组均可以明显增加K562/A02细胞的Caspase_3表达(P<0.05)。
结论
1.大蒜素DATS具有逆转K562/A02细胞耐药的作用,与VRP合用时具有协同作用。
2. DATS的这种逆转作用是通过减弱mdr-1基因的表达减少化疗药物外排和激活Caspase-3表达促进凋亡而实现的。
3. NF-κB通路参与了上述基因的调节过程。
Objectives
The multidrug resistance (MDR) of leukemic cells to chemotherapy remains the most significant cause of treatment failure in acute leukemia. A number of studies have shown that the major contributor to MDR is increased drug efflux mediated by P-glycoprotein (P-gp), a product of the mdr-1gene. Many plant-derived drugs or herbal formulations have been proved have anti-tumor potential in vitro and in vivo. Diallyl trisulfide (DATS) is the main sulfuric compound in garlic. Our goal was to explore whether diallyl trisulfide (DATS) could overcome P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in MDR K562/A02cells, and to prove whether NF-κB activation is involved in the MDR reversal mechanism of DATS.
Content and Methods
1. Assay of in vitro drug sensitivity
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay was used to compare the MDR of the K562and K562/A02cells to adriamycin. The absorbance value was measured with a spectrophotometer at the wavelength of570nm. IC50of the drugs was calculated based on the MTT assay. Inhibition of cell viability=(1-average A value of experimental group/average A value of blank control group)×100%. Drug resistance fold=IC50of drug-resistant group/IC50of sensitive group. Reverse fold=IC50before reversal/IC50after reversal
2. Observation of morphological changes by light microscopy
The morphological changes of apoptotic cells with Hematoxylin-Eosin (HE) staining were observed by light microscopy. K562/A02cells were grown at1×105cells/ml in a complete RPMI1640medium on a24-well plate with a coverslip set at the bottom, treatment with2μmol/L DATS combined with a final concentration of1 mg/L adriamycin for24h to72h,(treatment with adriamycin alone as control). Finally, the specimen was observed by light microscopy.
3. Detection of intracellular adriamycin concentration by FCM
The adriamycin concentration in K562/A02cells was measured by flow cytometry (FCM). K562/A02cells were treated with2μmol/L DATS or4mg/L verapamil and incubated for48h. A total of10000gated cells were detected for each sample, which were analyzed by Modfit LT software.
4. Detection of P-gp expression by FCM
P-gp expression on the surface membrane of K562/A02cells was determined by a direct immunoflourescence staining technique. K562/A02cells were co-cultured with2μmol/L DATS or4mg/L verapamil for48h. Protein expression was analyzed using Cell Quest software.
5.Apoptosis assay by statistical FCM
The apoptosis rates were measured using flow cytometric assay. Cell labeling was performed using annexin V conjugated to FITC, which binds to phosphatidylserine exposed on the surface membrane of cells undergoing apoptosis. The samples were measured using a flow cytometer with FACS software.
6.Semi-quantitative RT-PCR assay
After treatment with the drugs (2μmol/L DATS or4mg/L verapamil), in vitro total mRNA was extracted from the cells with Trizol reagent according to the manufacturer's instructions. The products were identified by electrophoresis using1.5%agarose gel. Using β-actin as internal reference, the products were further analyzed using Alpha gel image analysis system.
7.Western blot analysis of protein expression
After treatment with the drugs (2μmol/L DATS or4mg/L verapamil), total protein was isolated and subjected to sodium dodecyl sulfate PAGE analysis and transferred to a polyvinylidene difluoride membrane. After normalization by the corresponding β-actin expression, protein expression level was determined by densitometry scans and measured with Quantity One software.
8.Analyses
Statistical calculations were carried out with SPSS17.0for Windows software package. The results were expressed as mean±standard deviation of three independent experiments. Student's t-test was used for the statistical analyses, and P values<0.05were considered significant. The synergetic effect of the two drugs was analyzed using factorial analysis.
Results
1. Drug sensitivity
MTT assay was used to study the cytotoxicity of adriamycin. The ability of DATS at2μmol/L to enhance the cytotoxicity of adriamycin in K562/A02was examined. The IC50value of adriamycin for K562/A02decreased after treatment with DATS. The time-and concentration-dependent reversal effects of DATS on the K562/A02cells were observed for24,48, and72h. The higher the concentration of DATS used, the better the inhibitive effect.
2. Apoptosis observed by light microscopy
After simultaneous treatment of adriamycin (1μg/mL) with DATS (2μmol/L) for24h, the proliferation of K562/A02cells slowed down. After48h, K562/A02cells showed cell shrinkage, chromatin condensation, margination, nuclear fragmentation, apoptotic bodies, and typical apoptotic cytomorphological features. After72h, more apoptotic cells and less surviving K562/A02cells were detected.
3. Detection of intracellular adriamycin concentration
The effect of DATS on the intracellular accumulation of adriamycin was examined by FCM. After treatment with DATS, adriamycin fluorescence intensity in K562/A02cells increased to4.38±1.08, which there was a significant difference compared to that without DATS-treatment cells(P<0.01). These results showed that DATS had ability to enhance the intracellular concentration of adriamycin.
4. Alteration of P-gp expression
DATS-treated K562/A02cells were incubated with phycoerythrin-conjugated UIC2, and then detected by FCM. The expression of P-gp in K562was lower than that in K562/A02(P<0.01). After treatment with DATS or verapamil in K562/A02cells, P-gp expression decreased.
5. Apoptosis statistical FCM assay
Apoptosis of K562/A02cells was induced by DATS or verapamil. After incubation with either DATS (2μmol/L) or verapamil (4μg/ml) for48h, apoptotic percentages of K562/A02cells were12.15±0.78%and11.55±1.91%, respectively. Evident differences were found compared with the control (0.9±0.17%,P=0.000).
6. Detection of gene expression
As demonstrated by semi-quantitative RT-PCR, overexpression of mdrl mRNA was detected in K562/A02cells compared with K562cells (P=0.000). DATS could downregulate the expression of mdrl and NF-κB/p65(P<0.05) and upregulate the expression of caspase-3(P<0.05). However, DATS cannot evidently increase the expression level of IκBα, Bcl-2and Bax.(P>0.05)
7. Western blot analysis of protein expression
The Western blot report revealed that the expression of mdrl protein ai NF-KB/p65protein were much higher in K562/A02cells compared with K562ce (P=0.000). And the expression level of IκBα protein and caspase-3protein K562/A02cells was much lower than that of K562cells(P<0.05). DATS cou downregulate the expression of mdrl and NF-KB/p65(P<0.05) and upregulate t expression of caspase-3(P<0.05). However, DATS cannot evidently increase t expression level of IκBα(P>0.05)
Conclusion
The present study has demonstrated that DATS can serve as a novel, non-toxic modulator of MDR and can reverse the MDR of K562/A02cells in vitro by increasing intracellular adriamycin concentration, downregulating mdr-1expression, and inducing apoptosis by activating increased caspase-3expression. We therefore conclude that DATS can block NF-κB activation, which produces the downstream inhibitory effects on chemotherapy sensitivity and apoptosis of K562/A02cells. DATS could be a highly feasible candidate for the development of a new MDR reversal agent.
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