利用腺病毒载体过表达Meis 1基因能够上调化疗药对NSCLC细胞的杀伤作用

新型肿瘤抑制基因Meis 1(Myeloid ecotropic viral integration site 1)最近被证实可能具有肿瘤增殖抑制活性,是肿瘤患者预后和治疗的潜在指示分子,但其功能和作用机制尚需深入分析。考察利用腺病毒载体表达Meis 1蛋白对抗肿瘤药物杀伤NSCLC细胞的影响。非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞系A549感染Meis 1的腺病毒表达载体后,使用MTT、和软琼脂成集落实验验证其对NSCLC细胞增殖的抑制作用;进一步分别使用系列浓度梯度的抗肿瘤药物舒尼替尼(Sunitinib)、吉非替尼(Gefitinib)、紫杉醇(Paclitaxel)和吉西他滨(Gemcitabine)处理A549、Calu3、H460以及H358细胞,检测其抑制率,计算IC_(50)值。最后,利用耐药细胞系A549/ADR检测Meis 1逆转NSCLC化疗药多药耐药的作用。MTT和软琼脂成集落实验结果显示,Meis 1过表达能够抑制A549细胞的增殖与锚定非依赖性生长。抑制率实验显示Meis 1过表达能够上调抗肿瘤药物对NSCLC细胞的杀伤作用,显著下调其IC_(50)值。此外,Meis 1还具有逆转NSCLC细胞化疗药物多药耐药的作用。利用腺病毒载体表达MEIS1蛋白能够增加肿瘤细胞对化疗药物敏感型的作用,具有逆转肺癌细胞MDR的作用。

苯胺类毒物在非小细胞肺癌细胞中诱导肺耐药蛋白表达和抗肿瘤药物耐受

苯胺类毒物(aniline poison)是一类常用化工原料和环境污染物,通过食物或饮水污染进入体内后能够干扰人体正常内分泌系统(endocrinium)稳态,影响细胞遗传物质的稳定最终导致细胞DNA突变增加癌变或畸变的风险。最近的研究表明,苯胺类毒物还有可能影响恶性肿瘤的治疗效果。选取代表性的三个苯胺类毒物:对苯二胺(p-phenylene diamine,p-PDA)、间苯二胺(m-phenylene diamine,m-PDA)、邻苯二胺(o-phenylene diamine,o-PDA),考察其在非小细胞肺癌(non-small cell lung cancer,NSCLC)中诱导肺耐药蛋白(human lung resistance protein,LRP)表达及其可能的分子机制。培养NSCLC细胞系A549,使用系列浓度梯度的对苯二胺(p-phenylene diamine,p-PDA)、间苯二胺(m-phenylene diamine,m-PDA)、邻苯二胺(o-phenylene diamine,o-PDA)处理细胞后,使用实时荧光定量PCR(q PCR)检测LRP的mRNA表达;蛋白印记实验(Western bolt,WB)检测LRP的蛋白表达;进一步使用芳香烃受体(aryl hydrocarbon receptor,AhR)的小干扰RNA(siRNA)抑制A549细胞中AHR的表达,确定对苯二胺、间苯二胺、邻苯二胺对LRP的诱导作用是否依赖于AhR;在此基础上,使用对苯二胺、间苯二胺、邻苯二胺预处理A549细胞后,再分别使用抗肿瘤药物吉非替尼(Gefitinib)、吉西他滨(Gemcitabine)处理A549细胞,利用MTT实验检测上述抗肿瘤药物对A549细胞存活的抑制率(inhibitory rate,IR),并计算药物作用的半数抑制率(IC50值)。q PCR实验与WB实验结果显示,对苯二胺、间苯二胺、邻苯二胺刺激A549细胞能够剂量依赖地诱导LRP的mRNA、蛋白表达水平。干扰AhR表达后,该三种苯胺类毒物不能诱导LRP的mRNA与蛋白表达。MTT实验结果表明,对苯二胺、间苯二胺、邻苯二胺能够下调抗肿瘤药物吉非替尼、吉西他滨对A549细胞的杀伤作用:吉非替尼作用于A549细胞的IC50值从(1.20±0.25)μmol/L上调为(5.67±0.44)μmol/L、(6.07±0.30)μmol/L与(7.51±0.28)μmol/L,其耐药指数(resistance folds)分别为4.73、5.06与6.26;吉西他滨作用于A549细胞的IC_(50)值从(0.44±0.10)μmol/L上调至(1.55±0.25)μmol/L、(1.88±0.19)μmol/L与(2.33±0.40)μmol/L,其耐药指数分别为3.52、4.27与5.30。3种代表性苯胺类毒物能够在肺癌细胞中通过AhR诱导LRP表达,并且能够引起A549对抗肿瘤药物耐受。

脑胶质瘤细胞系U251中Nanog基因表达和细胞耐药及侵袭力的关系

目的探讨细胞耐药后干细胞因子Nanog表达及侵袭力的改变。方法脑胶质瘤细胞系U251卡莫司汀干预后通过RT-PCR检测干预前后细胞中Nanog在mRNA的水平表达情况,通过Transwell体外侵袭实验检测卡莫司汀干预前后侵袭力的改变。结果脑胶质瘤细胞系U251卡莫司汀干预5d后干细胞基因Nanog mRNA表达增高,卡莫司汀干预后穿膜细胞数高于干预前的细胞数(P<0.05)。结论卡莫司汀只能杀死普通肿瘤细胞,而肿瘤干细胞却相对增加,这是生长增殖、耐药和侵袭转移的主要原因。

The Expression of IMF-Kappa B Family Protein and Its Relationship with Drug Resistance in Breast Cancer Cell Lines

Objective: To study the expression of NFκB family protein in breast cancer cell lines and the relationship between NFκB family protein and the drug resistance. Methods: Expression of P65, IκB-α in 14 breast cancer cell lines and P50 in 11 breast cancer cell lines was detected by Western blot. The sensitivity of the cells to ADM was determined by MTT. Results: IκB-α located mainly in the cytoplasm. P65 and P50 were in both of cytoplasm and nucleus. The expression level of P50 was higher than that of P65, especially in nucleus. MTT assay showed that IC50 was three-fold higher in the cell lines which expressed P50 in nucleus than those P50 negative in nucleus, but no difference was found in the expression of P65. Conclusion: The expression of P50 in nucleus may predict the chemotherapy resistance in breast cancer, so it can be used as an indicator to predict the prognosis. The expression of P50 is more often in breast cancer, and it may play a more important role in the chemotherapy resistance than other NFκB family members.

The Expression of NF-Kappa B Family Protein and Its Relationship with Drug Resistance in Breast Cancer Cell Lines

Objective: To study the expression of NFκB family protein in breast cancer cell lines and the relationship between NFκB family protein and the drug resistance.Methods: Expression of P65, IκB-α in 14 breast cancer cell lines and P50 in 11 breast cancer cell lines was detected by Western blot. The sensitivity of the cells to ADM was determined by MTT.Results: 1κB-α located mainly in the cytoplasm. P65 and P50 were in both of cytoplasm and nucleus. The expression level of P50 was higher than that of P65, especially in nucleus. MTT assay showed that IC50 was three-fold higher in the cell lines which expressed P50 in nucleus than those P50 negative in nucleus, but no difference was found in the expression of P65.Conclusion: The expression of P50 in nucleus may predict the chemotherapy resistance in breast cancer, so it can be used as an indicator to predict the prognosis. The expression of P50 is more often in breast cancer, and it may play a more important role in the chemotherapy resistance than other NFκB family members. Key words breast cancer - NFκB - chemotherapy resistance

Use of arrays to investigate the contribution of ATP-binding cassette transporters to drug resistance in cancer chemotherapy and prediction of chemosensitivity

Multidrug resistance （MDR） is a major problem in cancer chemotherapy. One of the best known mechanisms of MDR is the elevated expression of ATP-binding cassette （ABC） transporters. While some members of human ABC transporters have been shown to cause drug resistance with elevated expression, it is not yet known whether the over-expression of other members could also contribute to drug resistance in many model cancer cell lines and clinics. The recent development ofmicroarrays and quantitative PCR arrays for expression profiling analysis of ABC transporters has helped address these issues. In this article, various arrays with limited or full list of ABC transporter genes and their use in identifying ABC transporter genes in drug resistance and chemo-sensitivity prediction will be reviewed.

ERK signaling pathway may induce gemcitabine chemoresistance in pancreatic cancer cell line SW1990 by regulating the expression of mdr-1 and RRM1 gene

Objective： To investigate the relationship between extracellular signal-regulated kinase （ERK） pathway, multidrug resistance gene （mdr-1）, ribonucleotide recluctase M1 （RRM1） gene and their roles in gemcitabine （GEM） chemoresistance in pancreatic cancer cell line SW1990. Methods： The GEM-resistance cell model was constructed by a stepwise method. Immunohistochemistry was used to measure the expression of ERK protein （ERK1/2） in the established cell strains in a semiquantitative way. The mRNA expression of mdr-1 and RRM1 were detected by RT-PCR. MTT assay was performed to determine the IC50 value. Results： The established GEM-resistant cell strains were able to gain stable growth and passage ability in the medium contained different concentration levels of GEM （0, 30, 60, 100, 150 and 200 nmol/L）. The expression of ERK protein, mdr-1 and RRM1 gene were elevated accompanied by the increase of GEM concentration. There was a highly positive correlation between mdr-1, RRM1 expression and GEM-resistanca level （r = 0.960, P = 0.002 and r = 0.966, P = 0.002）. The expression of ERK protein also correlated with the mdr-1 and RRM1 level （r = -0.943, P = 0.005 and r = -0.883, P = 0.02）. At the GEM-resistance level of 200 nmol/L, the grey scale value of ERK1/2 was 164.22 ±13.17, mdr-1/β-actin and RRM1/β-actin were 1.41 ±0.04 and 1.45 ± 0.18, respectively; after treated with ERK pathway inhibitor U0126, these values synchronously decreased to 186.85 ± 13.14, 0.2 3± 0.02 and 0.21 ± 0.03, respectively; inversely, the ERK1/2 grey scale value was 106.55 ± 16.45, mdr-l/β-actin and RRMl/β-actin were 1.50± 0.07 and 1.52 ± 0.12, respectively, which presented a tendency of synchronously increase after treated with ERK pathway activator EGF. The IC50 values in GEM-resistant cells of 0 nmol/L and 200 nmol/L levels were 4.104 and 10.20, respectively. After treated with U0126, these values decreased to 3.26 and 4.50, respectively; while treated with EGF, the IC50 values increased to 8.89 and 17.17, respectively. Conclusion： The ERK pathway may induce the GEM-chemoresistance in pancreatic cell line SW1990 through the participation in the regulation of the mdr-1 and RRM1 gene expression.

THE RELATIONSHIP BETWEEN mdrl GENE EXPRESSION AND DRUG-RESISTANCE IN OVARIAN CARCINOMA

Objective.To investigate the relationship between mdrl gene expression and multidrug-resistance in o- varian carcinoma. Design. We established tumor-bearing mice model of ovarian carcinoma,compared the anticancer drug sensitivity of OC/mdr-cell and OC/mdr+ cell in vitro, investigated the effect of cyclosporin A on reversing the multidrug resistance both in vitro and in the tumor-bearing mice model,detected the mdrl gene expre- sion in human ovarian carcinoma specimens. Main outcome measures. Anticancer drug sensitivity of both OC/mdr-cell and OC/mdr+ cell is mea- sured by the methods of MTT assays. mdrl gene expression is detected by the methods of RT-PCR. Results. Using MTT assay,OC/mdr+ cell is 4. 1 - 15. 5 times more resistant to VP-16,VCR,DNR, and DOX than OC/mdr-cell in vitro.2 μg/ml cyclosporine A(CsA)reduced the resistance of OC/mdr+ cell to DOX,from 0. 324±0. 072μg/ml to 0. 088±0. 024μg/ml. To OC/mdr－cell,CsA did not significantly in－ crease its sensitivity to DOX．Tumor-bearing mice with positive mdrl gene expression showed non-respon- siveness to DOX chemotherapy. When combined with intraperitoneal injection of CsA, the growth rate of tumor cells decreased significantly (P<0. 01 ). Only 4 of 23(17. 39% )tumors from patients who had not received chemotherapy exhibited positive mdrl gene expreession, while 6 of 9 (66. 67%)treated patients showed positive mdrl gene expression (Fisher exact test: P<0. 05 ). After cytoreductive surgery and chemotherapy, 14 of 19 untreated patients with negative mdrl gene expression had partial or complete re- sponse, while in patients with positive mdrl gene expression, 8 of 10 showed poor prognosis(Fisher exact test: P<0. 05 ). Conclusion. The expression of mdrl gene is associated with previous chemotherapy. CsA can reverse the resistance of mdr+ cells to indr-associated drs both in vitro and in vivo’ For the patients with ovarian carcinoma., the percentage of nonresponsiveness to the chemotherapy was found to be significantly higher among patients with positive mdrl gene expression than those with negative mdrl gene expression. mdrl gene expression can be detected to predict the clinical prognosis of patients with ovarian carcinoma.

Histone modification as a drug resistance driver in brain tumors

Patients with brain tumors,specifically,malignant forms such as glioblastoma,medulloblastoma and ependymoma,exhibit dismal survival rates despite advances in treatment strategies.Chemotherapeutics,the primary adjuvant treatment for human brain tumors following surgery,commonly lack efficacy due to either intrinsic or acquired drug resistance.New treatments targeting epigenetic factors are being explored.Post-translational histone modification provides a critical regulatory platform for processes such as chromosome condensation and segregation,apoptosis,gene transcription,and DNA replication and repair.This work reviews how aberrant histone modifications and alterations in histone-modifying enzymes can drive the acquisition of drug resistance in brain tumors.Elucidating these mechanisms should lead to new treatments for overcoming drug resistance.

Research progress in the use of combinations of platinum-based chemotherapy and epidermal growth factor receptor-tyrosine kinase inhibitors

In the past decade,the advent of the epidermal growth factor receptor-tyrosine kinase inhibitors(EGFR-TKIs)has dramatically influenced the therapeutic strategies for treating lung cancer,but with tumor progression and drug resistance,patients will ultimately develop reduced sensitivity to EGFR-TKIs.How can we delay the emergence of drug resistance? What is the next strategy after drug resistance? How to reasonably combine platinum-based chemotherapy and EGFR-TKIs? These questions are currently the focus of lung cancer research.Clinical studies have reported that platinum-based chemotherapy can increase the sensitivity to EGFR-TKIs.However,results of pre-clinical and clinical studies have been inconsistent.The mechanisms of platinum chemotherapy and EGFR-TKIs are still unknown due to the lack of systematic research.Therefore,systematic studies are required to show the mechanisms of EGFR-TKIs and chemotherapy agents and define the markers sensitive to their combinations when given concurrently or sequentially.

细胞凋亡抑制蛋白cIAP与卵巢癌耐药性的研究进展

卵巢恶性肿瘤是女性生殖系统三大恶性肿瘤之一，其发病率在女性生殖系统肿瘤中占第三位，而死亡率却高居首位。目前对于晚期卵巢癌（ⅡI或Ⅳ期）多倾向于用新辅助化疗＋肿瘤细胞减灭术＋术后周期性化疗的治疗方法。但是，尽管多数患者在最初对化疗药物较敏感，但仍有60％-80％最终死于卵巢癌，这些患者大部分都对化疗药物产生了耐药性，在更换新的化疗方案初期是有效的，但最终仍会耐药。近年来，有关细胞凋亡抑制蛋白（clAP，cellularinhibitorsofapoptosisproteins）在卵巢癌复发耐药中的作用机制的研究越来越受到重视。研究证实，clAP在耐药肿瘤细胞中呈高表达，并与多种因子共同参与形成了上皮性卵巢癌的耐药机制，抑制了化疗药物引起的肿瘤细胞的凋亡。这些发现为攻克卵巢癌的耐药机制提供了重要线索，也为卵巢癌化疗药物的应用指出了新的方向。

The regulation and functions of transcription factor Nrf2 in cancer chemoprevention and chemoresistance

Chemotherapy and chemoprevention have been two of the most important means to control cancer incidence and mortality, and the cellular defensive machinery against oxidative/electrophilic stresses plays significant roles in both means. This defensive system is composed of cytoprotective enzymes that metabolize and eliminate oxidative/electrophitic species. The transcription factor nuclear factor erythroid 2-related factor 2 （Nrf2） controls the basal and inducible expression of many cytoprotective genes, and plays a pivotal role in coordinating cellular defensive responses. Under basal conditions, the activity of Nrf2 is inhibited by binding to Kelch-like ECH-associated protein 1 （Keap 1）, which is capable of sensing oxidative/electrophilic signals. Upon oxidative/electrophilic stresses, the binding of Nrf2 to Keapl is disrupted, leading to activation of Nrf2 and induction of cytoprotective enzymes. Thus, Nrf2 has emerged as an important target of chemopreventive drugs. However, activation of Nrf2 could lead to very different outcomes depending on the cellular context. The indiscriminative protective effects of Nrf2 lead to its undesired functions in carcinogenesis and chemoresistance of cancer cells. Activation of Nrf2 provides neoplastic cells with growth advantages and protects cancer cells from chemotherapeutic drugs, resulting in poor clinical outcomes. In this means, inhibitors of Nrf2 signaling can enhance the efficacy of chemotherapeutic drugs and deserve further development. A better understanding of the regulation and functions of Nrf2 would be helpful for researches in both chemoprevention and chemotherapy of cancer.

MiR-1180 from bone marrow-derived mesenchymal stem cells induces glycolysis and chemoresistance in ovarian cancer cells by upregulating the Wnt signaling pathway

Background:Bone marrow-derived mesenchymal stem cells(BM-MSCs)play an important role in cancer development and progression.However,the mechanism by which they enhance the chemoresistance of ovarian cancer is unknown.Methods:Conditioned media of BM-MSCs(BM-MSC-CM)were analyzed using a technique based on microRNA arrays.The most highly expressed microRNAs were selected for testing their effects on glycolysis and chemoresistance in SKOV3 and COC1 ovarian cancer cells.The targeted gene and related signaling pathway were investigated using in silico analysis and in vitro cancer cell models.Kaplan-Merier survival analysis was performed on a population of 59 patients enrolled to analyze the clinical significance of microRNA findings in the prognosis of ovarian cancer.Results:MiR-1180 was the most abundant microRNA detected in BM-MSC-CM,which simultaneously induces glycolysis and chemoresistance(against cisplatin)in ovarian cancer cells.The secreted frizzled-related protein 1(SFRP1)gene was identified as a major target of miR-1180.The overexpression of miR-1180 led to the activation of Wnt signaling and its downstream components,namely Wnt5 a,β-catenin,c-Myc,and CyclinD1,which are responsible for glycolysis-induced chemoresistance.The miR-1180 level was inversely correlated with SFRP1 mRNA expression in ovarian cancer tissue.The overexpressed mi R-1180 was associated with a poor prognosis for the long-term(96-month)survival of ovarian cancer patients.Conclusions:BM-MSCs enhance the chemoresistance of ovarian cancer by releasing miR-1180.The released miR-1180 activates the Wnt signaling pathway in cancer cells by targeting SFRP1.The enhanced Wnt signaling upregulates the glycolytic level(i.e.Warburg effect),which reinforces the chemoresistance property of ovarian cancer cells.

Hollow iron oxide nanoparticles as multidrug resistant drug delivery and imaging vehicles

Magnetic nanopartides have been used as drug delivery vehicles against a number of cancer cells. Most of these theranostic formulations have used solid iron oxide nanoparticles （SIONPs） loaded with chemotherapeutics as nano-carrier formulation for both magnetic resonance imaging （MRI） and cancer therapy. In this study, we applied the dopamine-plus-human serum albumin （HSA） method to modify hollow iron oxide nanoparticles （HIONPs） and encapsuated doxorubicin （DOX） within the hollow porous structure of the nano-carrier. The new delivery system can load more drug than solid iron oxide nanoparticles of the same core size using the same coating strategy. The HIONPs-DOX formulation also has a pH-dependent drug release behaviour. Compared with free DOX, the HIONPs-DOX were more effectively uptaken by the multidrug resistant OVCAR8- ADR cells and consequently more potent in killing drug resistant cancer cells. MRI phantom and cell studies also showed that the HIONPs-DOX can decrease the T2 MRI signal intensity and can be used as a MR/contrast agent while acting as a drug delivery vehicle. For the first time, the dual application of chemo drug transport and MR imaging using the HIONPs-DOX formulation was achieved against both DOX-sensitive and DOX-resistant cancer cells.

卵巢癌的信息报道

卵巢癌是女性死亡率居第一位的恶性肿瘤。全世界每年约20万人确诊为卵巢癌,12.5万病人死于本病。尽管70%的病人初次化疗有效,可能达到缓解,但大多数终因对化疗药耐药而复发。我国卵巢癌每年新发病例5.2万,死亡2.2万。过去10年,发病率增长了30%,死亡率增长了18%。

MAFG-AS1/MAFG positive feedback loop contributes to cisplatin resistance in bladder urothelial carcinoma through antagonistic ferroptosis

Though promoting ferroptosis can reduce cisplatin resistance in tumor cells,ferroptosis and cisplatin resistance in bladder urothelial carcinoma(BUC)following long non-coding RNAs(lncRNAs)is largely unknown.Here,we found the highly expressed lncRNA MAF transcription factor G antisense RNA 1(MAFG-AS1)in BUC,and its inhibition increased the sensitivity of BUC cells to cisplatin by promoting ferroptosis.Mechanically,binding to iron chaperone poly(rC)-binding protein 2(PCBP2)facilitated the recruitments of MAFG-AS1 to deubiquitinase ubiquitin carboxyl-terminal hydrolase isozyme L5(UCHL5),thus stabilizing PCBP2 protein itself.Then PCBP2 was confirmed to interact with ferroportin 1(FPN1),an iron export protein,leading to inhibition of ferroptosis.Moreover,the expression of MAFG-AS1 was regulated by the transcriptional factor MAFG.Interestingly,MAFG-AS1 stimulated MAFG transcription by recruiting histone acetyltransferase p300(EP300)to promote the histone 3 at lysine 27(H3K27ac)at genomic locus of MAFG,forming a MAFG-AS1/MAFG positive feedback loop.In patient samples,higher expression of MAFG-AS1 and MAFG in BUC tissues was significantly correlated with T status and N status,such that MAFG-AS1,MAFG,and the combination of the two were independent prognostic indicators and chemotherapy sensitivity predictive biomarkers for BUC patients.These findings suggest that inhibition of MAFG-AS1 and MAFG can increase the sensitivity of BUC cells to cisplatin through promoting ferroptosis,indicating the novel chemotherapy sensitivity biomarkers and therapeutic target for BUC.