mTOR inhibitors sensitize thyroid cancer cells to cytotoxic effect of vemurafenib

Treatment options for advanced metastatic thyroid cancer patients are limited. Vemurafenib, a BRAFV600E inhibitor, has shown promise in clinical trials although cellular resistance occurs. Combination therapy that includes BRAFV600E inhibition and avoids resistance is a clinical need. We used an in vitro model to examine combination treatment with vemurafenib and mammalian target of rapamycin (mTOR) inhibitors, metformin and rapamycin. Cellular viability and apoptosis were analyzed in thyroid cell lines by trypan blue exclusion and TUNEL assays. Combination of vemurafenib and metformin decreased cell viability and increased apoptosis in both BCPAP papillary thyroid cancer cells and 8505c anaplastic thyroid cancer cells. This combination was also found to be active in vemurafenib-resistant BCPAP cells. Changes in expression of signaling molecules such as decreased mTOR expression in BCPAP and enhanced inhibition of phospho-MAPK in resistant BCPAP and 8505c were observed. The second combination of vemurafenib and rapamycin amplified cell death in BCPAP cells. We conclude that combination of BRAFV600E and mTOR inhibition forms the basis of a treatment regimen that should be further investigated in in vivo model systems. Metformin or rapamycin adjuvant treatment may provide clinical benefits with minimal side effects to BRAFV600E-positive advanced thyroid cancer patients treated with vemurafenib.     

米非司酮对人耐药卵巢癌细胞增殖、凋亡及其对紫杉醇敏感性的影响

背景与目的:米非司酮是有效的孕酮受体拮抗剂。研究发现,米非司酮对体内外卵巢癌细胞均具有生长抑制作用,但机制尚不清楚。本研究旨在探讨米非司酮对人耐药卵巢癌细胞A2780/T增殖、凋亡及其对紫杉醇敏感性的影响,为临床应用米非司酮治疗耐药性卵巢癌提供实验依据。方法:体外培养人卵巢癌耐药细胞A2780/T,采用CCK-8法检测单用米非司酮及合用紫杉醇时对A2780/T细胞增殖的影响,分析米非司酮与紫杉醇在抑制耐药卵巢癌细胞增殖中的相互作用。采用流式细胞术分析米非司酮及米非司酮联合紫杉醇对A2780/T细胞凋亡的影响。结果:实验所选各种浓度(0.625～20μg/mL)米非司酮对A2780/T和A2780细胞均有一定程度的生长抑制作用,并呈浓度依赖性。当紫杉醇浓度为1.25或2.5μg/mL,合用米非司酮浓度为20、10、5、2.5、1.25或0.625μg/mL时,能显著抑制A2780/T细胞的增殖,并显示两种药物的协同作用(q>1.15)。当紫杉醇浓度为0.625或5μg/mL时,仅表现为两种药物的相加作用(0.85相似文献   

VEGF反义寡核苷酸对Lewis肺癌细胞的抑制作用

目的:〖HT5"SS〗 探讨血管内皮生长因子（vascular endothelial growth factor, VEGF）反义寡核苷酸对C57BL/6小鼠肺癌细胞的抑制作用。〖HT5W〗方法:〖HT5"SS〗 制作C57BL/6小鼠皮下肺癌模型40只,分为VEGF反义寡核苷酸（ASODN）治疗组、VEGF正义寡核苷酸(SODN)治疗组、VEGF错义寡核苷酸（MODN）治疗组及对照组。接种Lewis肺癌细胞后24 h内,用ASODN、SODN及MODN皮下注射进行治疗,对照组只注射生理盐水,观察小鼠肿瘤的生长情况以及组织形态学改变,标本常规石蜡切片,HE染色,用免疫组化方法检测VEGF蛋白表达。〖HT5W〗结果:〖HT5"SS〗 对照组、ASODN组、SODN组、MODN组平均瘤重分别为（7.33±0.71）g、（4.56±0.38）g、(7.59±0.32)g和（7.62±0.39）g,ASODN组、SODN组、MODN组抑瘤率分别为43.8%、5.5%、3.1%。光镜下观察, VEGFASODN 能明显抑制肿瘤细胞生长,降低增殖活性。 免疫组化结果表明,ASODN组VEGF的表达水平明显低于SODN组、MODN组及对照组（P<0.05）。CD34免疫组化结果表明,ASODN组MVD为8.25±2.12,与对照组(14.78±3.51)、SODN组(13.71±3.62)及MODN组(12.81±2.56)比较,ASODN组MVD明显减少（P<0.01）。〖HT5W〗结论: 〖HT5"SS〗原位注射VEGF反义寡核苷酸能抑制小鼠肺癌生长。     

Antisense oligonucleotides to the epidermal growth factor receptor

Overexpression of the epidermal growth factor receptor (EGFR) has been observed in human breast tumors and is associated with poor prognosis in breast cancer patients. This would suggest that blocking the activity of the EGFR is a logical approach in the treatment of breast cancer. Three 20mer phosphorothioate oligodeoxynucleotides were designed to target different regions of the human epidermal growth factor receptor (EGFR) mRNA. Several analogs of these oligodeoxynucleotides (the 2fluoro analog, the 2propoxy analog, and/or the 5methyl cytosine analog) were also evaluated. We added these compounds to a human ovarian carcinoma cell line (SKOV3) and a human lung carcinoma line (A549), both of which overexpress the EGFR. All of these antisense oligonucleotides inhibited expression of the 10kb EGFR mRNA (range: 22–97% inhibition) compared to a scrambled control oligonucleotide or an untreated control. Expression of the less prominent 5.6kb EGFR mRNA band was also inhibited by all but two of the parent oligonucleotides. No inhibition of this 5.6kb band was found with the control oligonucleotide. The reduction in the expression of EGFR mRNA by the three most potent antisense compounds was accompanied by a significant reduction of EGFR protein (90–98%) and in vitro growth inhibition of SKOV3 cells as compared to the control oligonucleotide.     

Reversal of Taxol resistance in hepatoma by cyclosporin A: involvement of the PI-3 kinase-AKT 1 pathway

Hepatoma cells are known to be highly resistant to chemotherapy. Previously, we have found differential Taxol resistance in human and murine hepatoma cells. The aim of this study was to examine the effect of a multidrug resistance inhibitor, cyclosporin A in combination with Taxol on hepatoma in vitro and in vivo, and to identify the possible mechanism involved in Taxol resistance. Simultaneous treatment of cyclosporin A (0-10 microM) and Taxol (0.1 microM) inhibited cell growth in vitro. Cyclosporin A interfered with Taxol (0.1 microM)-induced AKT activation and BAD phosphorylation. Cyclosporin A combined with Taxol treatment augments caspase-9, -3 activation and loss of mitochondrial membrane potential in HepG2 cells. PI3 kinase inhibitor, wortmannin, or a dominant-negative AKT1 expression vector treatment partially enhanced Taxol-induced apoptosis indicating that PI3 kinase-AKT pathway was involved in Taxol-resistance pathway. Moreover, combination treatment reduced tumour growth in SCID and C57BL/6 mice as compared to either Taxol or cyclosporin A treatment. Our results indicate that the combination of cyclosporin A and Taxol is effective in the reversal of Taxol resistance through the inhibition of PI3 kinase-AKT1 pathway.     

泰素与卡铂联合治疗非小细胞肺癌的近期疗效观察

目的：探讨泰素对晚期非小细胞肺癌的疗效。方法：２６例晚期非小细胞肺癌应用泰素１３５ｍｇ／ｍ＾２加卡铂３００ｍｇ／ｍ＾２联合方案化疗。结果：１８例初治者和８例复治者近期有效率分别为６６．７％和５０％,总有效率６１．５％,有２例获ＣＲ。结论：泰素加卡铂对晚期非小细胞肺癌有效高疗效,特别是对治疗复发者也有较好的疗效。     

Mutational analysis of the class I beta-tubulin gene in human breast cancer

Non-small cell lung cancers have a high incidence of somatic mutations of the beta-tubulin (class I) gene, suggesting involvement in the acquisition of resistance to taxanes, which exert their effects through binding to beta-tubulin. Since taxanes are often used in the treatment of breast cancer, we carried out a mutational analysis of the class I beta-tubulin (GenBank accession AF070600) gene in breast cancer. We paid special attention to the primer design so as not to amplify the pseudogenes. We identified 1 somatic mutation, codon 306 [Arg (CGC) to Cys (TGC)], and 2 genetic polymorphisms, codon 217 [Leu (CTG) to Leu (CTA)] and (C to T) at 57 bases downstream from exon 4. Our results suggest that acquisition of resistance to taxanes is unlikely to be explained by somatic mutations of the class I beta-tubulin gene in most breast cancers. In addition, the overestimation of the incidence of somatic mutations of the class I beta-tubulin gene due to the pseudogenes is discussed.     

Bcl-x(L) antisense oligonucleotides induce apoptosis and increase sensitivity of pancreatic cancer cells to gemcitabine.

Pancreatic cancer is one of the leading causes of cancer-related death in Western countries. Bcl-x(L) is an anti-apoptotic factor of the Bcl-2 family, which is overexpressed in pancreatic cancer and its presence correlates with shorter patient survival. In this study, sequence-specific antisense oligonucleotides targeting the coding region of Bcl-x(L) were designed to examine whether apoptosis could be induced and chemosensitivity could be increased in pancreatic cancer cells. Five pancreatic cancer cell lines, Panc-1, MIA-PaCa-2, Capan-1, ASPC-1 and T3M4, were treated with Bcl-x(L) sense or antisense oligonucleotides and gemcitabine and the cell viability was examined by the SRB method. Apoptosis was determined using DAPI staining. In all examined pancreatic cancer cells, Bcl-x(L) expression was reduced after transfection of the antisense oligonucleotides. Cell death analysis using DAPI staining revealed that antisense, but not sense oligonucleotides caused apoptotic cell death. Furthermore, Bcl-x(L) antisense oligonucleotides enhanced the cytotoxic effects of gemcitabine in pancreatic cancer cells. Our results indicate that Bcl-x(L) antisense oligonucleotides effectively inhibited pancreatic cancer cell growth and caused apoptosis by reducing Bcl-x(L) protein levels. Bcl-x(L) antisense oligonucleotides also increased the chemosensitivity of pancreatic cancer cells, suggesting that Bcl-x(L) antisense therapy might be a potential future approach in this disease.     

c-jun反义核酸消除人体卵巢癌细胞的抗药性研究

目的:c-jun反义核酸消除人体卵巢癌细胞抗药性探讨.方法:采用c-jun反义核酸处理抗药型人体卵巢癌细胞,观察c-jun,γ-GCS以及GSH在两种细胞内的表达水平.结果:c-jun反义核酸可抑制c-jun基因的表达并降低谷胱苷肽合成的关键酶γ-GCS的mRNA表达量,使抗药型细胞内GSH含量降低大约75%.ATT分析结果也表明,经c-jun反义核酸处理后,抗药型细胞对顺铂的IC_(50)从40μmol/L降低到1.0μmol/L,对敏感型细胞无明显影响(0.2μmol/L).结论:c-jun反义核酸可特异地抑制抗药型细胞内c-jun基因的表达并间接抑制γ-GCS的表达,最终抑制GSH的合成,从而消除卵巢癌细胞的抗药性.     

XIAP反义寡核苷酸抑制胶质瘤细胞增殖和诱导凋亡的体外实验研究

目的观察X连锁凋亡抑制蛋白（XIAP）反义寡核苷酸对人脑胶质瘤细胞系BT325的增殖及凋亡的影响。方法将XIAP全硫代反义寡核苷酸，通过脂质体途径转染BT325脑胶质瘤细胞。用CCK-8实验检测细胞杀伤率，RT-PCR和Western blot技术检测XIAP的mRNA和蛋白表达，JC—1检测细胞线粒体膜电位，流式细胞仪检测细胞凋亡率，比色法检测Caspase-3活性。结果XIAP反义寡核苷酸能够抑制BT325脑胶质瘤细胞的增殖；XIAP反义寡核苷酸使XIAP的mRNA和蛋白表达均明显下调，mRNA较对照组减少了67．8％，蛋白较对照组减少了63．3％；反义寡核苷酸使线粒体膜电位下降，诱导BT325细胞凋亡，反义寡核苷酸组的凋亡率为54．7％，错义链组为14．O％，两组比较，差异具有显著性（P〈0．05）；反义寡核苷酸使BT325细胞Caspase-3活性明显增高，与对照组比较差异有显著性（P〈0．05）。结论XIAP可能参与了脑胶质瘤细胞BT325的增殖和凋亡，下调XIAP可抑制BT325细胞增殖及促进其凋亡。     

Effects of gemcitabine on APE/ref-1 endonuclease activity in pancreatic cancer cells, and the therapeutic potential of antisense oligonucleotides

Apurinic/apyrimidinic endonuclease (APE) is a key enzyme involved in DNA base excision repair (BER) that is often expressed at elevated levels in human cancers. Pancreatic cancer cells treated with the nucleoside analogue gemcitabine (2', 2'-difluoro-2'deoxycytidine) showed increases in APE/redox effector factor (ref-1) protein levels (approximately two-fold for Panc-1 and six-fold for MiaPaCa-2), with corresponding increases in endonuclease activity. These results suggested that the activation of APE/ref-1 might be an adaptive response that contributes to gemcitabine resistance by facilitating BER. To test this hypothesis, we examined the effects of disrupting APE/ref-1 using antisense on gemcitabine toxicity. Antisense oligonucleotides decreased protein levels three-fold in MiaPaCa-2 and five-fold in Panc-1 in comparison to controls, associated with reduced endonuclease activity. Combination treatments with antisense oligonucleotides and gemcitabine partially suppressed the increase in APE/ref-1 activity seen in cells exposed to gemcitabine alone. While clonogenic assays showed only slight decreases in colony formation in cells treated with either antisense oligonucleotides or gemcitabine alone, the combination with APE/ref-1 antisense resulted in a 2-log enhancement of gemcitabine toxicity in Panc-1 cells. Overall these findings suggest that APE/ref-1 plays a significant role in gemcitabine resistance in some pancreatic cancer cells, and support the further investigation of novel treatments that target this protein.     

外源MDR-1基因对小鼠肺腺癌细胞耐药性产生的影响及机理研究

目的 了解ＭＤＲ－１基因表达中细胞药性的关系。方法 用ＦＵＧＥＮ＾ＴＭ６转染试剂将ＭＤＲ－１基因导入小鼠Ｌｅｗｉｓ肺腺癌细胞株（３ＬＬ）,建立池耐药细胞株（３ＬＬ－ＭＤＲ－１）．用ＭＴＴ方法测定和划搏定对药的逆转效应,ＭＤＲ－１基因表达产物ｐ－ｇｐ采用免疫组化方法观察,应用流式细胞仪分析其对示踪剂罗丹明１２３的摄取与排泄。结果 建立了一株能稳定表达ｐ－ｇｐ和对长春生物碱类、鬼白纱类具有显著耐药性的     

A novel circular RNA,hsa_circ_0030998 suppresses lung cancer tumorigenesis and Taxol resistance by sponging miR‐558

Circular RNAs (circRNAs) are single‐stranded RNAs which form a covalently closed continuous loop. Although originally shown to be non‐protein‐coding, some circRNAs can give rise to micropeptides. circRNAs have also been shown to play essential regulatory roles in a variety of developmental and disease processes. In a previous study, hsa_circ_0030998 was identified as a circRNA downregulated in lung cancer, but its potential implications and mechanisms in lung cancer were not addressed. Here, we showed that overexpressing circ_0030998 decreased proliferation, migration, and invasion of lung cancer cells, while also dampening resistance to Taxol, a classical antitumor drug. Depleting circ_0030998 reversed these phenotypic effects. A high circ_0030998 expression was correlated with a high survival rate in lung cancer patients. Additionally, we found circ_0030998 could downregulate miR‐558 expression, serving as a microRNA sponge. In conclusion, our data support that hsa_circ_0030998 can slow down the progression of lung cancer by targeting miR‐558 and suppress malignant phenotypes such as proliferation, migration, and invasion progression of lung cancer cells. Therefore, we highlight that circ_0030998 could be a novel tumor suppressor of lung cancer.

Abbreviations

circRNA

circular RNA

IP

immunoprecipitation

LAMP1

lysosomal‐associated membrane protein 1

miRNA

microRNA

MMP

matrix metalloproteinase

NC

negative control

     

生存素反义寡核苷酸促进顺铂诱导骨肉瘤细胞凋亡

目的：探讨生存素（survivin）反义寡核苷酸（antisense oligonucleotide，ASODN）对顺铂（diamminedichloroplatinum，DDP）诱导骨肉瘤细胞凋亡的促进作用。方法：通过合成靶向survivin的ASODN转染骨肉瘤OS-732细胞并与DDP联合作用，同时设空白对照组、正义（SODN）组及DDP组进行比较。采用RT—PCR、免疫细胞化学法检测各组细胞survivin mRNA和蛋白表达状态，流式细胞仪（flow cytometry，FCM）、吖啶橙／溴化乙锭（acridine orange／ethidium bromide，AO／EB）染色法检测各组细胞凋亡水平和形态，MTT法检测细胞生长抑制情况。结果：与空白对照组、SODN组及DDP组相比，ASODN转染组细胞survivin mRNA及蛋白表达明显减弱，细胞凋亡水平较空白对照组、SODN组相应提高，细胞萎缩、染色质浓缩呈典型凋亡改变，细胞生长相对受抑；上述指标在ASODN＋DDP组变化更为明显。其细胞凋亡指数（apoptotic index，AI）和细胞生长抑制率（inhibition ratio，IR，61．36％）明显高于各单“药”组（SODN组6．81％、ASODN组31．82％和DDP组41．35％）及SODN＋DDP组（45．45％），P〈0．05。结论：As0DN能够特异性封闭骨肉瘤OS-732细胞中survivin基因表达，使其功能相应爱抑，增加细胞对DDP敏感性，进而增进DDP诱导骨肉瘤细胞凋亡效应。     

生存素反义寡核苷酸促进顺铂诱导骨肉瘤细胞凋亡

目的:探讨生存素(survivin)反义寡核苷酸(antisenseoligonucleotide,ASODN)对顺铂(diamminedichloroplatinum,DDP)诱导骨肉瘤细胞凋亡的促进作用。方法:通过合成靶向survivin的ASODN转染骨肉瘤OS732细胞并与DDP联合作用,同时设空白对照组、正义(SODN)组及DDP组进行比较。采用RTPCR、免疫细胞化学法检测各组细胞survivinmRNA和蛋白表达状态,流式细胞仪(flowcytometry,FCM)、吖啶橙/溴化乙锭(acridineorange/ethidiumbromide,AO/EB)染色法检测各组细胞凋亡水平和形态,MTT法检测细胞生长抑制情况。结果:与空白对照组、SODN组及DDP组相比,ASODN转染组细胞survivinmRNA及蛋白表达明显减弱,细胞凋亡水平较空白对照组、SODN组相应提高,细胞萎缩、染色质浓缩呈典型凋亡改变,细胞生长相对受抑;上述指标在ASODN+DDP组变化更为明显,其细胞凋亡指数(apoptoticindex,AI)和细胞生长抑制率(inhibitionratio,IR,61.36%)明显高于各单“药”组(SODN组6.81%、ASODN组31.82%和DDP组41.35%)及SODN+DDP组(45.45%),P<0.05。结论:ASODN能够特异性封闭骨肉瘤OS732细胞中survivin基因表达,使其功能相应受抑,增加细胞对DDP敏感性,进而增进DDP诱导骨肉瘤细胞凋亡效应。     

mrp反义RNA逆转胃癌细胞系SGC7901对VCR的耐受性

目的：探讨以多药耐药相关蛋白（ＭＲＰ）作为靶分子进行胃癌多药耐药基因治疗的可行性。方法：采用已构建成功的ｍｒｐ反义ＲＮＡ真核载体ｐｃＤＮＡ－Ａｍｒｐ转染胃癌细胞系ＳＧＣ７９０１,用Ｇ４１８抗性筛选出稳定细胞克隆,命名为Ｍ－ＳＧＣ７９０１;用＾３２Ｐ标记的寡核苷酸探针打点杂交检测Ｍ－ＳＧＣ７９０１细胞中ｍｒｐ ｍＲＮＡ表达的变化;从细胞生长曲线中,观察Ｍ－ＳＧＣ７９０１细胞生长速度的变化;经０．００     

泰素联合顺铂动、静脉给药治疗非小细胞肺癌的疗效比较

背景与目的：泰素（Taxol）联合顺铂（cisplatin,DDP）经静脉给药治疗晚期非小细胞肺癌（non-small cell lung cancer,NSCLC）,疗效较以往其他方案有所提高,但仍然不够理想。本研究的目的是比较晚期NSCLC患者经供瘤支气管动脉介入灌注化疗和经静脉化疗的临床疗效。方法：57例NSCLC患者随机分为靶动脉给药组（A组）和静脉给药组（B组）。A组27例,Taxol135mg/m^2,供瘤支气管动脉灌注3h,DDP80mg/m^2,动脉灌注1h,d1;B组30例,Taxol 135mg/m^2,DDP80mg/m^2,静脉点滴3h,d1。3-4周后重复,至少治疗2个周期。结果：近期疗效以CT或X光检查结果作为评价标准,CR1例,PR29例,总有效率52.63％。A组CR1例,PR17例,有效率为66.67％（18/27）;B组无CR,PR12例,有效率为40.00％（12/30）,两组有效率有显著性差异。A组中位疾病进展期（TTP）5.5个月,中位生存期10.5个月,0.5、1、2和3年生存率分别为85.18％、66.67％、48.15％和18.51％;B组TTP4.0个月,中位生存期10.5个月,0.5、1、2和3年生存率分别为70.00％、53.33％、30.00％和6.67％。不良反应主要为骨髓抑制、胃肠道反应和周围神经炎,经大量维生素、制酸、止呕、水化和应用升白细胞药物对症治疗后可缓解。结论：供瘤支气管动脉灌注Taxol和DDP是治疗晚期NSCLC的一种有效方法,供瘤支气管动脉给药后病灶局控率优于静脉注射。     

Antiproliferative effects of the histone deacetylase inhibitor FR901228 on small-cell lung cancer lines and drug-resistant sublines

FR901228 is a novel histone deacetylase (HDAC) inhibitor, and its antiproliferative effects on non-small cell lung cancer cells have been shown in vitro. However, there have been no reports concerning the effects on small-cell lung cancer (SCLC). We have recently demonstrated that the HDAC inhibitors trichostatin A and sodium butyrate inhibit expression of the catalytic subunit of telomerase (hTERT) mRNA and telomerase activity in prostate cancer cells. The present study was designed to evaluate the effects of FR901228 on proliferation and telomerase activity in SCLC cells in vitro. FR901228 at 5 to 10 nM increased the fraction of cells in the G(2)/M and sub-G(1) phases of the cell-cycle, and inhibited the growth of H69, H526 and H82 cell lines. The expression of hTERT mRNA was inhibited 6 hr after treatment, prior to obvious inhibition of cell growth or cell-cycle distribution shifts. The inhibition of hTERT mRNA expression and telomerase activity was not a consequence of cell-growth arrest or apoptosis. Cycloheximide blocked the suppression of hTERT mRNA induced by FR901228, and the inhibition of hTERT mRNA by FR901228 required newly synthesized proteins. FR901228 also effectively inhibited growth of etoposide-resistant UMCC-1/VP-16, irinotecan-resistant PC-6/SN2-5H and cisplatin-resistant H526/CDDP cells having decreased expression of hTERT mRNA and telomerase activity, as well as their parental cells. This implies that SCLC resistant to these key drugs are not cross-resistant to FR901228. The present study suggests that FR901228 may be a promising drug for chemotherapy of cancers including SCLC, even for refractory or relapsing tumors after conventional chemotherapy.