JNK1,JNK2,and JNK3 are involved in P-glycoprotein-mediated multidrug resistance of hepatocellular carcinoma cells

BACKGROUND:Multidrug resistance(MDR)is extremely common in hepatocellular carcinoma(HCC)and is a major problem in cancer eradication by limiting the efficacy of chemotherapy.Modulation of c-Jun NH2-terminal kinase(JNK)activation could be a new method to reverse MDR.However,the relationship between JNK activity and MDR in HCC cells is unknown.This study aimed to explore the relationship between MDR and JNK in HCC cell lines with different degrees of MDR.METHODS:A MDR human HCC cell line,SMMC-7721/ ADM,was developed by exposing parental cells to gradually increasing concentrations of adriamycin.The MTT assay was used to determine drug sensitivity.Flow cytometry was used to analyze the cell cycle distribution and to measure the expression levels of P-glycoprotein(P-gp)and MDR-related protein(MRP)-1 in these cells.JNK1,JNK2 and JNK3 mRNA expression levels were quantified by real-time PCR.Expression and phosphorylation of JNK1,JNK2,and JNK3 were analyzed by Western blotting.RESULTS:The MDR of SMMC-7721/ADM cells resistant to 0.05 mg/L adriamycin was mainly attributed to the overexpression of P-gp but not MRP1.In addition,these cells had a significant increase in percentage in the S phase,accompanied by a decrease in percentage in the G0/G1 phase,which is likely associated with a reduced ability for cell proliferation and MDR generation.We found that JNK1,JNK2,and JNK3 activities were negatively correlated with the degree of MDR in HCC cells.CONCLUSION:This study suggests that JNK1,JNK2,and JNK3 activities are negatively correlated with the degree of MDR in HCC cells.

Down-regulation of extracellular signal-regulated kinase 1/2 activity in P-glycoprotein-mediated multidrug resistant hepatocellular carcinoma cells

AIM： To study the expression and phosphorylation of extracellular signal-regulated kinase （ERK） i and ERK2 in multidrug resistant （MDR） hepatocellular carcinoma （HCC） cells.METHODS： MDR HCC cell lines, HepG2/adriamycin （ADM） and SMMC7721/ADM, were developed by exposing parental cells to stepwise increasing concentrations of ADM. MTT assay was used to determine drug sensitivity. Flow cytometry was employed to analyze cell cycle distribution and measure cell P-glycoprotein （P-gp） and multidrug resistant protein 1 （MRP1） expression levels. ERK1 and ERK2 mRNA expression lev-ls were measured by quantitative real-time PCR （QRTPCR）. Expression and phosphorylation of ERK1 and ERK2 were analyzed by Western blot.RESULTS： MTT assay showed that HepG2/ADM andSMMC7721/ADM were resistant not only to ADM, but also to multiple anticancer drugs. The P-gp expression was over 10-fold higher in HepG2/ADM cells than in HepG2 cells （8.92% ±0.22% vs 0.88% ± 0.05%, P 〈 0.001） and over 4-fold higher in SMMC7721/ADM cells than in SMMC7721 cells （7.37% ± 0.26% vs 1.74% ± 0.25%, P 〈 0.001）. However, the MRP1 expression was not significantly higher in HepG2/ADM and SMMC7721/ADM cells than in parental cells. In addition, the percentage of MDR HepG2/ADM and SMMC7721/ADM cells was significantly decreased in the G0/G1 phase and increased in the the S phase or G2/M phase. QRT-PCR analysis demonstrated that the ERK1 and ERK2 mRNA expression increased apparently in HepG2/ADM cells and decreased significantly in SMMC7721/ADM cells. Compared with the expression of parental cells, ERK1 and ERK2 protein expressions were markedly decreased in SMMC7721/ADM cells. However, ERK2 protein expression was markedly increased while ERK1 protein expression had no significant change in HepG2/ADM cells. Phosphorylation of ERK1 and ERK2 was markedly decreased in both HepG2/ADM and SMMC7721/ADM MDR cells.CONCLUSION： ERK1 and ERK2 activities are downregulated in P-gp-mediated MDR HCC cells. ERK1 or ERK2 might be a potential drug target for circumventing MDR HCC cells,

CIK cells from patients with HCC possess strong cytotoxicity to multidrug-resistant cell line Bel-7402/R

ABM: To investigate the cytotoxicity of the cytokine-induced killer (CIK) cells from the post-operation patients with primary hepatocellular carcinoma (HCC) to multidrug-resistant (MDR) cell of HCC both in vitro and in vivo. METHODS: A drug-resistant cell line was established by culturing human HCC cell line Bel-7402 in complete RPMI 1640 medium with increasing concentrations of adriamycin from 10 to 2 000 nmol/L. CIK cells were obtained by inducing the peripheral blood mononuclear cells with rhlFN-γ, monoclonal anti-CD3 antibody, rhIL-1α as well as rhIL-2, which were added into the culture. To detect the cytotoxicity of the CIK cells from HCC patients, the Bel-7402/R was taken as target (T) cells and CIK cells as effect (E) cells. Cytotoxic test was performed and measured by MTT. As to in vivo test, CIK cells were transfused into patients with HCC. The tumor specimens of the patients were obtained and immunohistochemistry was carried out to detect CD3, CD45, CD45RO as well as CD68. RESULTS: A MDR 1 HCC cell line Bel-7402/R was established. Its MDR1 mRNA overexpressed which was shown by RT-PCR; the P-glycoprotein expression increased from 1.32% of parent cells to 54%. CIK cells expanded vigorously by more than 70-fold and the CD3+CD56+ increased by more than 600-fold after 3-wk incubation on average. The cytotoxicity of CIK from HCC patients to Bel-7402/R was about 50% and to L-02 below 10% (t = 8.87, P<0.01), the same as that of CIK from normal individuals. Each of the 17 patients received 1-5×1010of CIK cell transfusion. No side effects were observed. After CIK treatment, the tumor tissue nodules formed and a large amount of lymphocytes infiltrated in the liver cancer tissue and CD3, CD45, CD45RO, and CD68 increased greatly which was shown by immunohistochemistry. CONCLUSION: A stable MDR1 HCC cell line has been established which could recover from liquid nitrogen and CIK from HCC patients has strong cytotoxicity to MDR HCC cell. CIK adoptive immunotherapy is safe and has no side effects. Receivers improved their immunity to tumor evidently. CIK treatment may be a better choice for HCC patients after operation to prevent the recurrence, especially when tumors have developed drug resistance.

原发性肝癌中Clusterin异常表达与多药耐药关系的研究

目的探讨聚集素(Clusterin)蛋白在原发性肝细胞癌(human hepatocellular carcinoma,HCC)中的表达及与多药耐药的关系。方法采用免疫组化S-P法检测41例原发性肝癌标本及10例正常肝组织、10例肝硬化组织中凋亡抑制基因Clusterin及Caspase-3、P-gp的表达。结果Clusterin在HCC中的阳性表达率为82.93%,在肝硬化和正常肝组织中表达阴性或弱阳性(82.93%vs10.00%χ2=28.89,P=0.00);Clusterin阳性表达与患者的年龄、性别、AFP值、HBsAg及肝硬化等无关,与Edmondson组织学分级有相关性。Caspase-3与P-gp在肝癌组织中的阳性表达率分别为34.15%和70.73%。肝癌组织中,Clusterin与Caspase-3表达呈负相关(r=-0.36,P=0.02),Clusterin与P-gp表达呈正相关(r=0.42,P=0.006)。结论Clusterin在HCC中呈过表达,与临床耐药密切相关,有望成为肝癌靶向治疗的一个新靶点;Clusterin通过直接或间接调控Caspase-3的活性从而抑制肝癌细胞凋亡,促进HCC的发生、发展及恶性增殖。

多药耐药真核表达载体的构建及其在人肝癌细胞HepG2中表达

目的 构建携带多药耐药mdr1全长基因的真核表达载体并研究其在人肝癌细胞HepG2 中的表达。方法 双酶切质粒pHaMDR1,获取含mdr1全长cDNA片断,将此片段定向克隆到真核表达载体PCI neo多克隆位点,经脂质体法转染HepG2细胞,G418筛选稳定的细胞系HepG2/mdr1,PCR检测mdr1 特异片段,RT PCR 检测HepG2/mdr1细胞mdr1 mRNA表达,流式细胞仪检测HepG2/mdr1 细胞P gp的含量。结果 成功构建携带mdr1全长基因的真核表达载体,并在HepG2 细胞中表达,形成稳定的细胞系HepG2/mdr1,其mdr1 mRNA及P gp的含量较未转染该载体的HepG2细胞显著增加。结论 用真核表达载体将mdr1 全长基因导入人肝癌细胞HepG2能够建立高效、稳定的多药耐药细胞系,为进一步研究多药耐药机理提供理想的细胞模型。

反义寡核苷酸降低细胞株SMMC-7721/ADM之MRP基因表达的研究

目的研究反义硫代磷酸寡核苷酸（ＡＳ－ＯＤＮ）抑制人肝癌细胞耐药株（ＳＭＭＣ－７７２１／ＡＤＭ）ＭＲＰ 基因表达的作用。方法用人工合成互补于ＭＲＰ 基因ｍＲＮＡ特定片断的反义硫代磷酸寡核苷酸，以脂质体Ｌｉｐｏｆｅｃｔａｍｉｎｅ为载体转染ＳＭＭＣ－７７２１／ＡＤＭ细胞株，用ＲＴ－ＰＣＲ测定ｍＲＮＡ表达，流式细胞技术测定细胞膜ＭＲＰ１蛋白Ｐ１９０表达及ＭＴＴ法检测细胞对柔红霉素（ＤＮＲ）和阿霉素（ＡＤＭ）的敏感性。结果 ＡＳ－ＯＤＮ能抑制 ＭＲＰｍＲＮＡ和其蛋白 Ｐ１９０表达，提高细胞对 ＤＮＲ和 ＡＤＭ的敏感性。结论① ＡＳ－ＯＤＮ能降低 ＭＲＰ基因表达；② ＭＲＰ介导的多药耐药（ ＭＤＲ）是ＳＭＭＣ－７７２１／ＡＤＭ耐药的重要机理之一。

晚期糖基化终产物对人肝癌细胞HepG2耐药性的影响

目的:探讨晚期糖基化终产物(advanced glycation end products,AGEs)对肝癌细胞耐药性的影响及其机制。方法:体外培养人肝癌细胞HepG2,以终浓度分别为100、200、400μg.ml-1的AGEs联合2 000 nmol.L-1盐酸阿霉素(adramycin,ADM)处理细胞24 h,并设空白对照和ADM组进行比较。应用倒置显微镜观察不同浓度AGEs联合ADM孵育下HepG2形态学变化,流式细胞术(flow cytometry,FCM)检测细胞周期的改变,应用细胞计数试剂盒(cell counting kit-8,CCK-8)检测细胞株活性,蛋白质免疫印迹法(Western blotting)测定不同浓度AGEs作用下,HepG2细胞多药耐药基因(multidrug resistance-1,MDR1)蛋白表达情况。结果:在2 000 nmol.L-1ADM作用下,HepG2细胞生长停滞或死亡,而AGEs能促进细胞生长,抑制其死亡。细胞周期和细胞活性实验表明,与ADM组相比,随着AGEs浓度增加,G1期细胞百分率显著下降,S期及G2期细胞增加,细胞活性有上升趋势。Western blotting检测表明AGEs能增加MDR1的蛋白表达。结论:AGEs能通过促进MDR1基因的表达,降低肿瘤细胞对化疗药物的敏感性。

多药耐药真核表达载体的构建及其生物学特性

目的构建携带多药耐药相关蛋白(MRP)全长基因的真核表达载体,并研究其在人肝癌细胞株HepG2中的表达特性。方法双酶切克隆质粒pGEM-mrp1,获得含mrp1全长的cDNA片段,再将此片段定向克隆到哺乳动物真核表达载体pCI-neo的多克隆位点,经脂质体法转染HepG2细胞,G418筛选获得稳定表达的转基因细胞系HepG2/mrp1。RT-PCR检测HepG2/mrp1细胞mrp1 mRNA表达,流式细胞仪检测HepG2/mrp1细胞MRP的含量。结果本实验所构建的携带mrp1全长基因的表达载体pCI-mrp1能在HepG2细胞中表达,形成稳定的多药耐药细胞系HepG2/mrp1,其多药耐药性、MRP含量及mrp1 mRNA表达均较未转染该载体的HepG2细胞显著增加。结论将携带全长人多药耐药相关蛋白基因mrp1的载体导入人肝癌细胞株能够建立高效、稳定的多药耐药细胞株,为进一步研究多药耐药的机理及逆转多药耐药提供理想的细胞模型。

人肝癌Bel7402/5-FU耐药株多药耐药与凋亡关系的研究

目的 从凋亡的角度探讨人肝癌细胞Bel74 0 2 / 5 FU多药耐药的分子机制。方法 采用SRB法检测Bel74 0 2 / 5 FU耐药株的多药耐药性 ,用AnnexinV FITC/PI双标流式细胞术检测Bel74 0 2细胞及其耐药株的凋亡程度 ,PI单染检测细胞周期变化 ,RT PCR方法检测凋亡相关因子bcl 2、bcl xl、bcl xs、bax、fas、p5 3和cpp32的表达情况。 结果 Bel74 0 2 / 5 FU耐药株具有多药耐药性 ;相同剂量 30 μmol·L-1及 15 0 μmol·L-15 FU处理 ,Bel74 0 2细胞凋亡明显增加 ,G0 /G1期增加 ,S期、G2 /GM减少 ,耐药株不出现凋亡增加及细胞周期改变 ;耐药株凋亡相关因子bcl xl、bcl xs和bax表达上调 ,p5 3和cpp32表达下调。 结论 人肝癌Bel74 0 2 / 5 FU耐药株的多药耐药性可能和凋亡减少有关 ,抗凋亡因子bcl xl表达上调 ,促凋亡因子 p5 3和cpp32表达下调可能参与Bel74 0 2 / 5 FU耐药株的多药耐药。

单基因人肝癌耐药细胞株（HepG2／MRP1）的构建及其生物学意义

目的对抗肿瘤药的天然耐药和化疗过程中产生的继发性耐药是肝癌化疗敏感性差的重要原因之一，为探讨体外逆转肝癌多药耐药（multidrug resistance，MDR）的新方法，笔者建立了单因素人肝癌细胞多药耐药模型HepG2／mrp1，并研究其生物学特性。方法双酶切克隆质粒pGEM—mrp1获得人全长多药耐药相关蛋白基因（mrp1），并将其插入哺乳动物表达载体pCI—neo的多克隆位点，构建重组载体，利用质脂体法将重组载体转人人肝癌细胞HepG2，建立单基因耐药细胞株HepG2／mrp1。观察细胞的生长规律；MTT法检测其多药耐药性；流式细胞仪检测细胞表面多药耐药相关蛋白（multidrug resistance-associated protein，MRP）的表达；RT—PCR检测MRP1 tuRNA表达量。结果与HepG2细胞相比较，HepG2／mrp1细胞的倍增时间延长30．86h。该细胞对多种抗肿瘤药耐药，HepG2／mrp1对阿霉素和柔红霉素的耐药指数比亲本细胞增加了11．4倍和8倍，细胞表面MRP1的表达明显增加，mrp1 tuRNA明显增加。结论HepG2／mrp1细胞稳定高表达MRP1，具有多药耐药性，为进一步研究肝癌的多药耐药构建了一个技术平台。

HepG2/mdr1稳定细胞系的建立及特性研究

将已构建好的含有人多药耐药(multidrug resistance,MDR)全长基因的真核表达质粒pCI-neo-mdr1,应用脂质体导入人肝癌HepG2细胞,应用G418筛选出人肝癌多药耐药细胞株HepG2/mdr1。通过对HepG2/mdr1细胞形态学的观察和生物学特性的研究,成功地建立了高效、稳定的HepG2/mdr1细胞系;为深入研究肝癌的MDR及其逆转提供了理想的细胞模型,并为探索建立肝癌MDR细胞株提供新的方法和思路,同时为研究肝癌细胞胰岛素抵抗与MDR的关系提供了模型细胞。