Nucleophosmin与肝癌细胞多药耐药性关系的初步研究
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摘要
背景
原发性肝癌是世界常见的恶性肿瘤之一,全球约半数的肝癌患者集中在中国。目前临床肝癌诊疗中还存在着早期诊断困难、复发转移率高,预后差等特点,其死亡率在我国恶性肿瘤中居第二位。在肝癌的综合治疗中,化疗是重要方法之一,而肝癌的多药耐药已成为制约化疗效果及影响患者生存的主要难点。Nucleophosmin (NPM)是一核仁磷酸化蛋白,它参与RNA合成与装配、染色体复制,调节抑癌基因p53和p14的活性,作为分子伴侣防止蛋白集聚,在细胞增殖和生长中发挥重要作用。现有研究报道,NPM可能是胃癌、结肠癌、卵巢癌和前列腺癌的肿瘤标记物之一,而且NPM可能通过抑制依赖于干扰素调节因子(IRF-1)的抗癌监视作用参与肿瘤的发生发展。Polo-like kinase 1 (Plkl)属于有丝分裂丝氨酸／苏氨酸激酶家族,是G2／M期DNA损伤检测点重要激酶,对中心体的成熟,双极纺锤体的形成及胞浆分裂等起重要作用。Plk1作为启动细胞分裂和正性推进的酶,在细胞增殖过程中起重要作用。在多种肿瘤中,Plk1常表现为过表达并与不良预后密切相关。有研究报道,靶向Plk1的反义寡核苷酸可以增强乳腺癌细胞对多西他赛的敏感性。因此,我们设想NPM及Plk1在肝癌的多药耐药中也发挥类似作用。
目的
1、观察肝癌多药耐药细胞中NPM的表达,并应用其特异性抑制剂NSC348884下调其表达,明确NPM是否参与肝癌多药耐药；
2、利用基因工程技术转染NPM至HepG2,建立稳定转染细胞系,探讨NPM在肝癌多药耐药中的作用；
3、检测Plk1在HepG2/NPM的表达,探讨其在NPM介导肝癌多药耐药的作用。
方法
1、检测肝癌细胞株HepG2及耐药株HepG2/ADM中NPM的蛋白及mRNA表达量；应用NPM特异性抑制剂NSC348884,观察其对HepG2/ADM耐药性的影响：(1)CCK-8法检测NSC348884对HepG2/ADM细胞的剂量—效应曲线；(2)CCK-8法检测NSC348884预处理耐药细胞后,应用三种不同机制化疗药物(ADM,5-FU和DDP)后的细胞存活率；(3)罗丹明蓄积实验检测对细胞膜上p-gP功能的影响。
2、构建稳定转染细胞系HepG2/NPM,上调NPM的表达,观察对化疗药物敏感性的影响：(1)CCK-8法检测应用三种化疗药物后转染细胞的存活率；(2)流式细胞术AnnexinV-PI法检测对5-Fu的凋亡率；(3) Caspase-3检测细胞的凋亡；(4) Western blot检测多药耐药相关基因蛋白的表达；(5) real-time PCR检测多药耐药相关基因的表达。
3、检测Polo-like kinase 1在HepG2和HepG2／NPM的表达,使用Polo-like kinase 1特异性抑制剂BI2536,观察对转染细胞多药耐药性的影响,进行如下实验：(1)Polo-like kinase1蛋白及mRNA表达的检测；(2)CCK-8法检测BI2536对HepG2/NPM细胞的剂量-效应曲线；(3)CCK-8法检测BI2536预处理后应用ADM,5-Fu及DDP对HepG2/NPM存活率的影响；(4)Annexin V-PI检测经过B12536预处理后的HepG2/NPM转染细胞系对5-Fu的凋亡率；(5)Western blot检测多药耐药相关基因蛋白的表达；(6)real-time PCR检测多药耐药相关基因的表达。
结果
1、肝癌多药耐药细胞(HepG2/ADM)中NPM的高表达
经Western blot及real-time PCR检测,发现耐药细胞HepG2/ADM与亲代细胞HepG2相比NPM蛋白和mRNA均明显上升,有统计学意义,说明这种上升发生于基因水平。给予Nucleophosmin特异性抑制剂NSC348884预处理HepG2/ADM,细胞对ADM、DDP、5-Fu三种化疗药物的敏感性增强。
2、稳定转染细胞系的建立及耐药性检测
采用LipofectamineTM 2000转染pEGFP/NPM至肝癌细胞HepG2, G418筛选,建系。采用real-time PCR检测稳定转染细胞NPM mRNA表达水平,Western blot检测蛋白表达,均可以发现NPM明显上调,说明稳定转染细胞系HepG2/NPM成功建立。
CCK-8法结果显示,HepG2/NPM于ADM、DDP和5-Fu的敏感性均显著低于亲本细胞HepG2(p<0.05),说明NPM增强了HepG2的多药耐药性。与CCK-8法结果一致,AnnexinV-PI双标法检测发现HepG2／NPM给药后凋亡率下降,细胞呈现出对化疗药物的耐受性。与HepG2组相比,HepG2/NPM组Caspase-3相对活性显著降低。Western blot结果显示：多药耐药相关基因中MDR-1和MRP-1的蛋白表达水平较对照组明显升高,而LRP-1的表达无明显变化。real-time PCR结果显示在HepG2/NPM中MDR-1和MRP-1表达明显增高,而LRP-1的表达无明显变化,提示NPM介导的肝癌细胞多药耐药可能与MDR-1和MRP-1的表达上调有关,这种改变发生于基因水平。
3、Polo-like kinase 1 (Plkl)在NPM参与肝癌MDR中的作用
Plk1在HepG2/NPM较HepG2蛋白表达明显上升,而mRNA表达无差异,说明改变发生于转录后而非基因水平。经BI2536作用后,HepG2/NPM细胞对各药的敏感性增强,其IC50明显下降(P<0.05)。结果显示,BI2536处理后再给予5-Fu,细胞凋亡率上升,说明敏感性增强。Western blot结果显示：给予BI2536处理后HepG2/NPM细胞MDR-1蛋白含量显著下降,而MRP-1及LRP-1蛋白含量无明显变化。real-time PCR检测发现给药后HepG2/NPM细胞MDR-1的mRNA水平降低,提示Plk-1对MDR-1的作用发生于基因水平。
结论
1、NPM增强了肝癌细胞HepG2的多药耐药性；
2、NPM介导的肝癌细胞多药耐药性与其抑制Caspase-3的活性和对MDR-1及MRP-1的上调有关,而与LRP-1无关；
3、Plk-1参与了NPM介导的肝癌MDR,其机制可能与其对MDR-1基因表达的上调有关。
Background
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, and about half of the patients with liver cancer focused in China, where about 50,000 people die from liver cancer each year, ranking the second in malignant tumor mortality. Chemotherapy is one of the important ways in the comprehensive treatment for liver cancer, while the tumor multidrug resistance(MDR) is one of the main reasons for significant effects on chemotherapy and patients' survival. Nucleophosmin (NPM, also known as B23, numatrin,or NO38), was first identified as a nucleolar phosphoprotein expressed at high levels in the granular region of the nucleolus. NPM was soon thought to have a role in the regulation of cell growth, proliferation and transformation, based on the observation that its expression rapidly increases in response to mitogenic stimuli, and that increased amounts of the protein are detected inhighly proliferating and malignant cells. Another of the mechanisms through which NPM might suppress apoptosis is the functional inhibition of p53 and p14. The NPM protein is overexpressed in various tumours, and it has been proposed as a marker for gastric, colon, ovarian and prostate carcinomas. Polo-like kinase 1(Plkl) is a serine/threonine kinase.Many studies have shown that the Plkl has multiple functions during mitosis and,perhaps more importantly, has a significant role in ensuring the fidelity of checkpoint controls. with non-small-cell lung cancer. Multiple follow-on studies showed that determining Plkl expression levels has prognostic value for different cancers, including oropharyngeal carcinoma, melanoma, colon cancer and hepato-blastoma. Furthermore, some reports have indicated that Plkl expression is a reliable marker for identifying a high risk of metastasis.lt is reported that antisense inhibitors against Plkl may be considered as highly efficient promoters for the antineoplastic potential of taxanes, causing synergistic effects in breast cancer cells.Thus, we postulated that Plkl has the same role in MDR of HCC.
Objective
1.To investigate the expression of NPM in HepG2/ADM and study the the effects of NPM on multidrug resistance of HCC;
2.To construct a recombinant plasmid carrying enhanced green fluorescent protein and human NPM gene and transfected into HepG2, with the aim of better elucidating the mechanisms of NPM in MDR of HCC;
3.To investigate the expression of Plkl in HepG2/NPM,with the aim of better elucidating the mechanisms of Plkl in MDR of HCC.
Methods:
1.The expressions of NPM in HepG2/ADM and HepG2 were determined by real-time PCR and Western blot.
2.The effects of NSC348884, which is a special inhibitor of NPM, on MDR of HepG2/ADM were examined by following perspects:
(1) By CCK-8, the dose-response curve of NSC348884 on HepG2/ADM cell was drawn, and with the flow cytometry to detect the effect of NSC348884 with above-mentioned dose on Rh123 concentration in HepG2/ADM cell, then, the appropriate dose of NSC348884 was selected according to the above experimental results.
(2) Cell proliferation were determined by Cell counting Kit-8
(3) Rh 123 accumulation is used to detect the effect of NSC348884 with above-mentioned dose in HepG2/ADM cell, then, the appropriate dose of NSC348884 was selected according to the above experimental results.
3.The recombinant plasmid pEGFP/NPM was transfected into HepG2 with lipofectamineTM2000. It was divided into two groups:HepG2 cell and HepG2/NPM cell group:
(1)Detected cell proliferation by Cell counting Kit-8;
(2)Potentiation of drug-induced apoptosis by NSC348884 was evaluated by using Annexin V/propidium iodide flow cytometry;
(3)Caspase-3 activity assay was used to analyze apoptosis of cells.
(4)The protein expression level of MDR-1,MRP-1 and LRP-1 were determined by Western blot;
(5)The gene expression level of MDR-1,MRP-1 and LRP-1 were determined by real-timePCR.
4.The effects of BI2536,which is a special inhibitor of Plkl, on MDR of HepG2/NPM were examined by following perspects:
(1)The expressions of Plkl in HepG2/NPM and HepG2 were determined by real-time PCR and Western blot;
(2)By CCK-8, the dose-response curve of BI2536 on HepG2/NPM cell was drawn, the appropriate dose of BI2536 was selected according to the above experimental results;
(3)Detected cell proliferation by Cell counting Kit-8;
(4)Potentiation of drug-induced apoptosis by BI2536 was evaluated by using Annexin V/ Propidium iodide flow cytometry;
(5)The protein expression level of MDR-1,MRP-1 and LRP-1 were determined by Western blot;
(6)The gene expression level of MDR-1 was determined by real time-PCR.
Results:
1.The levels of NPM in HepG2/ADM
1.1 Compared with HepG2 cells,NPM was identified that show significantly high expression in HepG2/ADM cells by real-time PCR and Western blot analysis.
1.2The results showed treatment with NSC348884 for 24h followed by ADM,5-Fu or DDP for 48 hours resulted in significant cell growth inhibition.
2.Establishment of HepG2/NPM
The recombinant plasmid carrying enhanced green fluorescent protein and human NPM gene was successfully constructed and transfected into HepG2.The protein was secreted which confirmed by real-time PCR and Western blot.
2.1 Measurement of MDR in HepG2/NPM
2.1.1CCK-8 assay reveals that overexpression of NPM produced multidrug resistance (10.08 folds of ADM-resistance,7.16 folds of 5-Fu-resistance and 2.98 folds of DDP-resistance).
2.1.2The combination of NSC348884 and 5-Fu resulted in a stronger apoptotic effect in comparison with either agent alone after 48h treatment by using Annexin V/propidium iodide flow cytometry (P<0.05).
2.1.3To further test the ability of NPM on apoptosis, caspase-3 in both cell lines tested were evaluated.It is revealed that NPM caused a decrease in caspase-3 activity (P<0.05).
2.1.4Effects of NPM on MDR-1,MRP-1 and LRP-1 expression
The MDR-1 and MRP-1 protein level in HepG2/NPM was greatly increased as compared to that of HepG2(4.93±0.60 vs 1.12±0.21和2.49±0.35 vs 1.12±0.21, P<0.05). Whereas there was no significant change in LRP-1 protein level among them(1.13±0.09 vs 1.11±0.08, P>0.05).
2.1.5Changes of MDR-1,MRP-1 and LRP-1 mRNA level
Consistent with the results of protein level,real-time PCR showed MDR-1 and MRP-1 expression mRNA wasere markedly increased in HepG2/NPM than in HepG2.
3.The role of Plkl in MDR nduced by NPM of HepG2
3.1 Compared with HepG2 cells,Plkl was identified that show significantly high expression in HepG2/NPM cells by real-time PCR and Western blot analysis;
3.2 the role of Plkl in MDR induced by NPM of HepG2
3.2.1 To all these three chemotherapy drugs-ADM, DDP and 5-Fu, HepG2/NPM cell showed resistance. Under the action of BI2536, the sensitivity of the drug-resistant cell HepG2/NPM enhanced to all the anticancer drugs, the IC5o decreased significantly (P<0.05).
3.2.2The combination of BI2536 and 5-Fu resulted in a stronger apoptotic effect in comparison with either agent alone after 48h treatment by using Annexin V/propidium iodide flow cytometry (P<0.05).
3.2.3Effects of NPM on MDR-1,MRP-1 and LRP-1 expression
Our results of western blot displayed the MDR-1 expression in HepG2/NPM was significantly up-regulated after BI2536 treatment. Whereas there was no significant change in MRP-1 and LRP-1 protein level.
3.2.4Changes of MDR-1 mRNA level
To determine whether the change in MDR-1 protein expression inHepG2/NPM is regulated at a transcriptional level, mRNA level of MDR-1 was analyzed by a real time-PCR assay. There were no significant changes in MDR-1 mRNA level among all groups.
Conclusions
1.Overexpression of NPM could induce MDR in HepG2 cells, indicating that NPM plays an important role in the drug-resistant phenotype of human hepatic cancer cells;
2.The role of NPM in MDR of HCC is associated with MDR-1 and MRP-1;
3.Plk1 is involved in MDR of HepG2 induced by NPM.
原发性肝癌是世界常见的恶性肿瘤之一,全球约半数的肝癌患者集中在中国。目前临床肝癌诊疗中还存在着早期诊断困难、复发转移率高,预后差等特点,其死亡率在我国恶性肿瘤中居第二位。在肝癌的综合治疗中,化疗是重要方法之一,而肝癌的多药耐药已成为制约化疗效果及影响患者生存的主要难点。Nucleophosmin (NPM)是一核仁磷酸化蛋白,它参与RNA合成与装配、染色体复制,调节抑癌基因p53和p14的活性,作为分子伴侣防止蛋白集聚,在细胞增殖和生长中发挥重要作用。现有研究报道,NPM可能是胃癌、结肠癌、卵巢癌和前列腺癌的肿瘤标记物之一,而且NPM可能通过抑制依赖于干扰素调节因子(IRF-1)的抗癌监视作用参与肿瘤的发生发展。Polo-like kinase 1 (Plkl)属于有丝分裂丝氨酸／苏氨酸激酶家族,是G2／M期DNA损伤检测点重要激酶,对中心体的成熟,双极纺锤体的形成及胞浆分裂等起重要作用。Plk1作为启动细胞分裂和正性推进的酶,在细胞增殖过程中起重要作用。在多种肿瘤中,Plk1常表现为过表达并与不良预后密切相关。有研究报道,靶向Plk1的反义寡核苷酸可以增强乳腺癌细胞对多西他赛的敏感性。因此,我们设想NPM及Plk1在肝癌的多药耐药中也发挥类似作用。
目的
1、观察肝癌多药耐药细胞中NPM的表达,并应用其特异性抑制剂NSC348884下调其表达,明确NPM是否参与肝癌多药耐药；
2、利用基因工程技术转染NPM至HepG2,建立稳定转染细胞系,探讨NPM在肝癌多药耐药中的作用；
3、检测Plk1在HepG2/NPM的表达,探讨其在NPM介导肝癌多药耐药的作用。
方法
1、检测肝癌细胞株HepG2及耐药株HepG2/ADM中NPM的蛋白及mRNA表达量；应用NPM特异性抑制剂NSC348884,观察其对HepG2/ADM耐药性的影响：(1)CCK-8法检测NSC348884对HepG2/ADM细胞的剂量—效应曲线；(2)CCK-8法检测NSC348884预处理耐药细胞后,应用三种不同机制化疗药物(ADM,5-FU和DDP)后的细胞存活率；(3)罗丹明蓄积实验检测对细胞膜上p-gP功能的影响。
2、构建稳定转染细胞系HepG2/NPM,上调NPM的表达,观察对化疗药物敏感性的影响：(1)CCK-8法检测应用三种化疗药物后转染细胞的存活率；(2)流式细胞术AnnexinV-PI法检测对5-Fu的凋亡率；(3) Caspase-3检测细胞的凋亡；(4) Western blot检测多药耐药相关基因蛋白的表达；(5) real-time PCR检测多药耐药相关基因的表达。
3、检测Polo-like kinase 1在HepG2和HepG2／NPM的表达,使用Polo-like kinase 1特异性抑制剂BI2536,观察对转染细胞多药耐药性的影响,进行如下实验：(1)Polo-like kinase1蛋白及mRNA表达的检测；(2)CCK-8法检测BI2536对HepG2/NPM细胞的剂量-效应曲线；(3)CCK-8法检测BI2536预处理后应用ADM,5-Fu及DDP对HepG2/NPM存活率的影响；(4)Annexin V-PI检测经过B12536预处理后的HepG2/NPM转染细胞系对5-Fu的凋亡率；(5)Western blot检测多药耐药相关基因蛋白的表达；(6)real-time PCR检测多药耐药相关基因的表达。
结果
1、肝癌多药耐药细胞(HepG2/ADM)中NPM的高表达
经Western blot及real-time PCR检测,发现耐药细胞HepG2/ADM与亲代细胞HepG2相比NPM蛋白和mRNA均明显上升,有统计学意义,说明这种上升发生于基因水平。给予Nucleophosmin特异性抑制剂NSC348884预处理HepG2/ADM,细胞对ADM、DDP、5-Fu三种化疗药物的敏感性增强。
2、稳定转染细胞系的建立及耐药性检测
采用LipofectamineTM 2000转染pEGFP/NPM至肝癌细胞HepG2, G418筛选,建系。采用real-time PCR检测稳定转染细胞NPM mRNA表达水平,Western blot检测蛋白表达,均可以发现NPM明显上调,说明稳定转染细胞系HepG2/NPM成功建立。
CCK-8法结果显示,HepG2/NPM于ADM、DDP和5-Fu的敏感性均显著低于亲本细胞HepG2(p<0.05),说明NPM增强了HepG2的多药耐药性。与CCK-8法结果一致,AnnexinV-PI双标法检测发现HepG2／NPM给药后凋亡率下降,细胞呈现出对化疗药物的耐受性。与HepG2组相比,HepG2/NPM组Caspase-3相对活性显著降低。Western blot结果显示：多药耐药相关基因中MDR-1和MRP-1的蛋白表达水平较对照组明显升高,而LRP-1的表达无明显变化。real-time PCR结果显示在HepG2/NPM中MDR-1和MRP-1表达明显增高,而LRP-1的表达无明显变化,提示NPM介导的肝癌细胞多药耐药可能与MDR-1和MRP-1的表达上调有关,这种改变发生于基因水平。
3、Polo-like kinase 1 (Plkl)在NPM参与肝癌MDR中的作用
Plk1在HepG2/NPM较HepG2蛋白表达明显上升,而mRNA表达无差异,说明改变发生于转录后而非基因水平。经BI2536作用后,HepG2/NPM细胞对各药的敏感性增强,其IC50明显下降(P<0.05)。结果显示,BI2536处理后再给予5-Fu,细胞凋亡率上升,说明敏感性增强。Western blot结果显示：给予BI2536处理后HepG2/NPM细胞MDR-1蛋白含量显著下降,而MRP-1及LRP-1蛋白含量无明显变化。real-time PCR检测发现给药后HepG2/NPM细胞MDR-1的mRNA水平降低,提示Plk-1对MDR-1的作用发生于基因水平。
结论
1、NPM增强了肝癌细胞HepG2的多药耐药性；
2、NPM介导的肝癌细胞多药耐药性与其抑制Caspase-3的活性和对MDR-1及MRP-1的上调有关,而与LRP-1无关；
3、Plk-1参与了NPM介导的肝癌MDR,其机制可能与其对MDR-1基因表达的上调有关。
Background
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, and about half of the patients with liver cancer focused in China, where about 50,000 people die from liver cancer each year, ranking the second in malignant tumor mortality. Chemotherapy is one of the important ways in the comprehensive treatment for liver cancer, while the tumor multidrug resistance(MDR) is one of the main reasons for significant effects on chemotherapy and patients' survival. Nucleophosmin (NPM, also known as B23, numatrin,or NO38), was first identified as a nucleolar phosphoprotein expressed at high levels in the granular region of the nucleolus. NPM was soon thought to have a role in the regulation of cell growth, proliferation and transformation, based on the observation that its expression rapidly increases in response to mitogenic stimuli, and that increased amounts of the protein are detected inhighly proliferating and malignant cells. Another of the mechanisms through which NPM might suppress apoptosis is the functional inhibition of p53 and p14. The NPM protein is overexpressed in various tumours, and it has been proposed as a marker for gastric, colon, ovarian and prostate carcinomas. Polo-like kinase 1(Plkl) is a serine/threonine kinase.Many studies have shown that the Plkl has multiple functions during mitosis and,perhaps more importantly, has a significant role in ensuring the fidelity of checkpoint controls. with non-small-cell lung cancer. Multiple follow-on studies showed that determining Plkl expression levels has prognostic value for different cancers, including oropharyngeal carcinoma, melanoma, colon cancer and hepato-blastoma. Furthermore, some reports have indicated that Plkl expression is a reliable marker for identifying a high risk of metastasis.lt is reported that antisense inhibitors against Plkl may be considered as highly efficient promoters for the antineoplastic potential of taxanes, causing synergistic effects in breast cancer cells.Thus, we postulated that Plkl has the same role in MDR of HCC.
Objective
1.To investigate the expression of NPM in HepG2/ADM and study the the effects of NPM on multidrug resistance of HCC;
2.To construct a recombinant plasmid carrying enhanced green fluorescent protein and human NPM gene and transfected into HepG2, with the aim of better elucidating the mechanisms of NPM in MDR of HCC;
3.To investigate the expression of Plkl in HepG2/NPM,with the aim of better elucidating the mechanisms of Plkl in MDR of HCC.
Methods:
1.The expressions of NPM in HepG2/ADM and HepG2 were determined by real-time PCR and Western blot.
2.The effects of NSC348884, which is a special inhibitor of NPM, on MDR of HepG2/ADM were examined by following perspects:
(1) By CCK-8, the dose-response curve of NSC348884 on HepG2/ADM cell was drawn, and with the flow cytometry to detect the effect of NSC348884 with above-mentioned dose on Rh123 concentration in HepG2/ADM cell, then, the appropriate dose of NSC348884 was selected according to the above experimental results.
(2) Cell proliferation were determined by Cell counting Kit-8
(3) Rh 123 accumulation is used to detect the effect of NSC348884 with above-mentioned dose in HepG2/ADM cell, then, the appropriate dose of NSC348884 was selected according to the above experimental results.
3.The recombinant plasmid pEGFP/NPM was transfected into HepG2 with lipofectamineTM2000. It was divided into two groups:HepG2 cell and HepG2/NPM cell group:
(1)Detected cell proliferation by Cell counting Kit-8;
(2)Potentiation of drug-induced apoptosis by NSC348884 was evaluated by using Annexin V/propidium iodide flow cytometry;
(3)Caspase-3 activity assay was used to analyze apoptosis of cells.
(4)The protein expression level of MDR-1,MRP-1 and LRP-1 were determined by Western blot;
(5)The gene expression level of MDR-1,MRP-1 and LRP-1 were determined by real-timePCR.
4.The effects of BI2536,which is a special inhibitor of Plkl, on MDR of HepG2/NPM were examined by following perspects:
(1)The expressions of Plkl in HepG2/NPM and HepG2 were determined by real-time PCR and Western blot;
(2)By CCK-8, the dose-response curve of BI2536 on HepG2/NPM cell was drawn, the appropriate dose of BI2536 was selected according to the above experimental results;
(3)Detected cell proliferation by Cell counting Kit-8;
(4)Potentiation of drug-induced apoptosis by BI2536 was evaluated by using Annexin V/ Propidium iodide flow cytometry;
(5)The protein expression level of MDR-1,MRP-1 and LRP-1 were determined by Western blot;
(6)The gene expression level of MDR-1 was determined by real time-PCR.
Results:
1.The levels of NPM in HepG2/ADM
1.1 Compared with HepG2 cells,NPM was identified that show significantly high expression in HepG2/ADM cells by real-time PCR and Western blot analysis.
1.2The results showed treatment with NSC348884 for 24h followed by ADM,5-Fu or DDP for 48 hours resulted in significant cell growth inhibition.
2.Establishment of HepG2/NPM
The recombinant plasmid carrying enhanced green fluorescent protein and human NPM gene was successfully constructed and transfected into HepG2.The protein was secreted which confirmed by real-time PCR and Western blot.
2.1 Measurement of MDR in HepG2/NPM
2.1.1CCK-8 assay reveals that overexpression of NPM produced multidrug resistance (10.08 folds of ADM-resistance,7.16 folds of 5-Fu-resistance and 2.98 folds of DDP-resistance).
2.1.2The combination of NSC348884 and 5-Fu resulted in a stronger apoptotic effect in comparison with either agent alone after 48h treatment by using Annexin V/propidium iodide flow cytometry (P<0.05).
2.1.3To further test the ability of NPM on apoptosis, caspase-3 in both cell lines tested were evaluated.It is revealed that NPM caused a decrease in caspase-3 activity (P<0.05).
2.1.4Effects of NPM on MDR-1,MRP-1 and LRP-1 expression
The MDR-1 and MRP-1 protein level in HepG2/NPM was greatly increased as compared to that of HepG2(4.93±0.60 vs 1.12±0.21和2.49±0.35 vs 1.12±0.21, P<0.05). Whereas there was no significant change in LRP-1 protein level among them(1.13±0.09 vs 1.11±0.08, P>0.05).
2.1.5Changes of MDR-1,MRP-1 and LRP-1 mRNA level
Consistent with the results of protein level,real-time PCR showed MDR-1 and MRP-1 expression mRNA wasere markedly increased in HepG2/NPM than in HepG2.
3.The role of Plkl in MDR nduced by NPM of HepG2
3.1 Compared with HepG2 cells,Plkl was identified that show significantly high expression in HepG2/NPM cells by real-time PCR and Western blot analysis;
3.2 the role of Plkl in MDR induced by NPM of HepG2
3.2.1 To all these three chemotherapy drugs-ADM, DDP and 5-Fu, HepG2/NPM cell showed resistance. Under the action of BI2536, the sensitivity of the drug-resistant cell HepG2/NPM enhanced to all the anticancer drugs, the IC5o decreased significantly (P<0.05).
3.2.2The combination of BI2536 and 5-Fu resulted in a stronger apoptotic effect in comparison with either agent alone after 48h treatment by using Annexin V/propidium iodide flow cytometry (P<0.05).
3.2.3Effects of NPM on MDR-1,MRP-1 and LRP-1 expression
Our results of western blot displayed the MDR-1 expression in HepG2/NPM was significantly up-regulated after BI2536 treatment. Whereas there was no significant change in MRP-1 and LRP-1 protein level.
3.2.4Changes of MDR-1 mRNA level
To determine whether the change in MDR-1 protein expression inHepG2/NPM is regulated at a transcriptional level, mRNA level of MDR-1 was analyzed by a real time-PCR assay. There were no significant changes in MDR-1 mRNA level among all groups.
Conclusions
1.Overexpression of NPM could induce MDR in HepG2 cells, indicating that NPM plays an important role in the drug-resistant phenotype of human hepatic cancer cells;
2.The role of NPM in MDR of HCC is associated with MDR-1 and MRP-1;
3.Plk1 is involved in MDR of HepG2 induced by NPM.
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